Tracking Cell Lineages of Single Cells
Entry by Andrew Capulli, AP225 Fall 2011
"Tracking lineages of single cells in lines using a microfluidic device" Amy C. Rowat, James C. Bird, Jeremy J. Agresti, Oliver J. Rando, and David A. Weitz, Proc. Natl. Acad. Sci. U. S. A. 106(43), 18149–18154 (2009).
While some cell lines continue to express apparently similar phenotypes over time as they replicate, other cell populations don't replicate at all (or do so very minimally). Take the human body in say, ten years of aging: skin tissue and the cells therein are relatively conserved, meaning the skin you have at the starting point will look and will physically be very similar to your skin in 10 years. Cardiac myocytes, those that make up your heart tissue will also 'look' the same but for a different reason: for the most part, they are the same (these are very general statements but they illustrate the point that follows). Cell expression (protein expression) as a function of time and generation is a somewhat under investigated area. As generally mentioned about tissues, the behaviors of cells in bulk (tissue) can and have been observed but the behaviors of individual cells and cells lines are masked in the three dimensional "ensemble" as the authors call it. In tissues or even just unconstrained clusters of cells (say, in a petri dish of culture flask) the lineages of dividing cells become mixed in the three dimensional space so when staining and investigation into particular phenotypes (protein expression) is investigated, it may be clear which cells are expressing the phenotype but their 'age' or 'generation' is unclear... tracking if the cells are newly spawned is impossible. Therefore, in these simple set-ups it is impossible to track phenotype as it varies down one lineage (conserved genetic makeup) of cell replication (starting with one cell). Here the authors propose to address this question with a novel microfluidic device that allows them to investigate the phenotype of a single cell lineage via separate chambers that house a single cell. The chambers allow for the cell to replicate down the chamber(1 dimensionally) and consequently the authors, after a period of replications, have the consequent generations starting from a single cell assembled in a line where they can then investigate cell phenotype.
Lineage Chamber Device
Much of this paper describes the lineage chamber device as shown below in Figure 1 from the paper. A detailed account is in the paper but I'll now summarize the device for the purpose of discussion later on. Thin chambers (1 budding yeast, Saccharomyces cerevisiae, cell wide) are assembled in parallel (50 chambers in parallel per unit). The chamber can be seen in part A of Figure 1. Cells and media are injected into the device; as the authors describe, once a single cell occupies a chamber, the flow in that chamber is reduced and consequently it is less likely that another cell flows into that chamber. The flow through the device is described by the previous wiki entry on this paper that can be found by clicking: http://soft-matter.seas.harvard.edu/index.php/Tracking_lineages_of_single_cells_in_lines_using_a_microfluidic_device. Essentially, via the bypass flow paths between chambers, the authors achieve a 70% 'seeding' of a single cell per chamber which only takes about 3 minutes; the remaining 30% of the chambers may have clusters of cells or no cells. The chamber geometry is such that it allows the cell to enter upstream of media flow but does not allow the cell to exit via a constriction at the downstream exit of the chamber. The chamber dimensions are designed based on the average cell size of the yeast cell, in theory this only allows for cell replication upstream in one dimension; this means the original cell will remain downstream while subsequent generations will be spawned upstream but are not free to leave the chamber due to the incoming flow that keeps them in.
Figure 2 from the paper is more revealing of how the device works. The arrows in Part A of Figure 2 shows the direction of flow of the cell media. Notice the single cell in the chamber on the right; the geometry of the chamber in addition to the flow constrains the cell to this position and future generations to positions in the chamber upstream. Part B shows that there remains flow within the trapping chamber with or without a trapped cell; however, once a cell is trapped in the chamber, flow increases in the bypass which, as the authors note, decreases the possibility of another cell entering that chamber. This is the novelty of the device: loading of single cells into chambers without manual manipulation increases efficiency of the study beyond previous capabilities (this can be done, as mentioned, in 3 minutes). Flow based loading, although not 100% accurate as seen in Part D of Figure 2, is quite successful (70% success, which is better efficiency than predictions made by Poisson statistics (40%)). Taking a step back for a minute, we can appreciate the previous difficulties this device has overcome: cells are very small and the manual manipulation of a single cells is rather difficult. This device loads a single cell into a chamber that is only the width of a single cell (this is what I would consider impossible or close to impossible via manual manipulation or even robotics given the soft and nonuniform nature of cells).
The thumb image on the right (from Figure 3 of the paper) shows the lineage of single cell over time up the chamber.
Phenotype As a Function Generation: Paper Results
As discussed above, the inquiry into how cell phenotype or expression varies as a function of generation within a single cell lineage is poorly understood because of the complex nature of addressing the question: a single cell needs to be isolated and allowed to replicate and its progeny need to be organized so we can essentially identify "who's who" or which is the original cell and which are the progeny and how old they are. The authors, as summarized in the previous section, have developed a device for such a set up via the use of microfluidics and what I've termed "flow loading" of individual cell chambers. The expression level (phenotype) of three proteins was investigated. pPho84-GFP expression (a high-affinity phosphate transporter) was found to be maintained among the generations; as the authors note, this protein is found to be 'switched' either fully on or off in a cell and consequently switched on or off in progeny. The authors found this to be the case; in generations of a single cell, all expressed or none expressed this protein. However, the heat shock protein Hsp12-GFP was found to be expressed at various levels among the generations which suggests that the phenotype of the protein is not conserved among the generations and may be dictated by other factors. The "housekeeping" protein as the authors call it, Rps8b-GFP was found to be completely conserved and expressed throughout the generations; this is a ribosomal protein needed for the cell to function so these results are to be expected. The results of the three proteins are seen in Figure 4b below: