Tracking lineages of single cells in lines using a microfluidic device

From Soft-Matter
Revision as of 19:45, 17 September 2010 by Amao (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

Entry by Angelo Mao, AP 225, Fall 2010

Title: Tracking lineages of single cells in lines using a microfluidic device

Authors: Amy C. Rowat, James C. Bird, Jeremy J. Agresti, Oliver J. Rando, David A. Weitz

Journal: Proceedings of the National Academy of Sciences

Volume: 106(43)

Pages: 18149-18154


Phenotypes vary down generations, but the time scale of variation in relation to cell division has not been elucidated because research platforms have been unable to isolate single cells to track their generations. The researchers created an in vitro microfluidics device that could trap single cells and observe their and their progeny's protein expression. The researchers demonstrated that the device could observe protein expression in real time, and applied this technique to three proteins that varied in expression across generations. Though the mechanisms are unclear, this research showed that phenotypic variation occurred to different degrees even in progeny of a single parent cell.

Soft Matter Keywords: microfluidics, epigenetics, in vitro

Device Fabrication

Fig 1. Schematic depicting microfluidic channels. D to A depict the layout in increasing magnification.

To trap single cells, researchers designed channels (Fig 1A) with dimensions large enough for one cell, but constricted at the end so as not to let the cell escape. To prevent clogging of the channel, the researchers included bypass channels and calculated the expected flow rate through expected channels by modeling the system in terms analogous to electric circuits and by using the Hagen-Poiseuille relation.

<math> \frac{Q_{2}}{Q_{1}} = \frac{R_{1a} + R_{1b}}{R_{2}} = \frac{h_{2}^3 w_{2}}{l_{2}} (\frac{l_{1a}}{h_{1a}^{3} w_{1a}} + \frac{l_{1b}}{h_{1b}^{3} w_{1b}}) </math>

Here, the ratio describes the volumetric flow rate ratio of the bypass channel to the trapping channel. The variables h, w, and l stand for height, width and length.

Results and Conclusion

The researchers used yeast as model eukaryote cells to test their device. Since a constant nutrient source was necessary for cell viability, the researchers ensured by using fluorescent markers that, even in channels where cells were trapped, cell media was available.

Fig 2. Results of protein expression. The long vertical line of cells at the right depict cells in the channel after numerous divisions. Vertical lines in the graphs on the left depict lineages, and brightnesses correlate to the brightness of protein expression.

Researchers tagged threeproteins of interest, pPho84, the heat shock protein Hsp12 and the ribosomal protein Rps8b, with fluorescent markers. Real time imaging was taken of the single cell as it divided. Whereas Rps8b stayed fairly constant, Hsp12 experienced fluctuations, represented by the blue, yellow or red rectangles on the graph to the left. The relative consistency of fluorescence was quantified to obtain a "cluster index," which is the number of adjacent cells with similar fluorescence divided by the total number of cells (Fig 3). The variation of expression are demonstrated to be different for these two proteins, but the researchers did not quantify the degree of that difference.

Fig 3. Cluster index of the fluorescently tagged pPho84 protein on left, and fluorescently tagged Hsp12 on the right. CI closer to 1 indicates greater degree of similarity.

Although the mechanism behind variations in protein expression in cells derived from a common parent cell, this platform allows for study of this phenomenon.