This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.
Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.
Budding yeast is a powerful model organism in aging research. However, a conventional lifespan assay in yeast relies on microdissection, which is not only labor intensive but also low throughput1,2. In addition, the traditional microdissection approach does not provide a detailed view of various cellular and molecular features in the single mother cells as they age. The development of microfluidic devices has enabled an automated procedure to measure yeast lifespan as well as to follow molecular markers and various cellular phenotypes throughout the lifespan of the mother cells3,4,5,6,7,8. After yeast cells are loaded into a microfluidic device, they can be tracked under a microscope using automatic time-lapse imaging. With the help of imaging processing tools, various cellular and molecular phenotypes can be extracted3,6,8, including lifespan, size, fluorescent reporter, cell morphology, cell cycle dynamics, etc., many of which are difficult or impossible to obtain using the traditional microdissection method. Microfluidic devices have gained prominence in yeast aging research since their successful development a few years ago3,4,6,7. Several groups have subsequently published on variations of the earlier designs5, and many yeast labs have employed microfluidic devices for their study.
In a cell culture undergoing exponential growth, the number of aged mother cells that are available for observation is miniscule. Therefore, the general design principle of the microfluidic device for lifespan measurements is to retain mother cells and to remove daughter cells. One such designs makes use of the fact that yeast undergoes asymmetric cell division. The structures in the device will trap bigger mother cells and allow smaller daughter cells to be washed away. The microfluidic chip described in this article uses a soft polydimethylsiloxane (PDMS) pad (vertical pensile columns) to trap mother cells (Figure 1). Devices of similar design have been reported previously3,4,6,7. This protocol uses a simpler procedure to fabricate microfluidic devices and a straightforward cell-loading method that is optimized for the time-lapse imaging experiments. One of the key parameters in the microfluidic device is the width of the PDMS pads used to trap mother cells. Our device uses wider pads that can keep more mother cells under each pad, including a significant fraction of fresh mother cells that can be tracked throughout their lifespan. In addition to lifespan measurements, this protocol is useful for single cell time-lapse imaging experiments when the cells need to be tracked for many generations or when an observation throughout the entire lifespan is necessary.
1. Silicon Wafer Mold Fabrication
NOTE: The photomask is designed with AutoCAD software and manufactured by a commercial company. This design contains three layers of different patterns (Supplementary File 1). The heights of the first, second, and third layers are about 4 µm, 10 µm, and 50 µm, respectively. The silicon wafer mold was created from the photomask using soft lithography9,10.
2. Microfluidic Chip Fabrication
3. Preparing for the Experiment
After the experiments, the lifespans of the cells and many cellular and molecular phenotypes can be extracted from the recorded time-lapse images. Since there are a number of different features that can be extracted from each cell, the first step of the analysis is to annotate the cells and events, including the positions and boundaries of the cells and the timing of various events that are being tracked, such as the budding events. These annotations will make it easier to return to the same set of cells and analyze different features in the future. Using image analysis software, such as ImageJ11 and the associated plugins, a list of phenotypes can then be extracted from the image data using the recorded annotations of the cells.
The following are a few examples of phenotypes measured with the microfluidic device. The lifespan phenotype of yeast can be obtained by counting the number of buds produced by each trapped mother cell and estimating with the Kaplan-Meier estimator. The cells initially trapped in the microfluidic devices are of unknown ages. To minimize the bias of lifespan measurements originating from these unknown ages, previous methods used the average number of bud scars on the trapped cells to calibrate the lifespan4,7. However, bud scar measurements require the additional staining of cells and provide only an indirect estimation of the bias. Using this protocol, the device frequently traps fresh daughter cells that budded off from already trapped cells. These cells then turn into mother cells. Such cells can be identified using our downstream image analysis software (Movie 1). These fresh mother cells provide a more accurate lifespan measurement. As shown in Figure 2, the lifespan curves of fresh mother cells were compared with those of the cells of unknown age. In this experiment, the average lifespan of the fresh mother cells was slightly longer (about 2 generations of difference in median lifespan).
In addition to the number of buds, other interesting phenotypes, such as the time interval between two successive budding events, as shown in Figure 3, can be extracted from the imaging data3,7,8. Cells entered a period of faster budding following a few slower initial buddings. The budding slowed down dramatically towards the end of their lifespan, indicating the unhealthy state of these aged cells. The cell cycle dynamics contains very useful information regarding the cellular state of the young and old cells and has been used, for example, to characterize telomerase mutants12. Importantly, this device can be used to measure fluorescence signals throughout the lifespan (Figure 4), allowing for the tracking of molecular markers that may be the driver of the aging process.
Figure 1: A schematic of the design of the microfluidic device. The device consists of 6 independent functional modules that can operate in parallel. Each module is made of one main channel connected to two side channels. Each side channel has 113 pensile columns. An additional bridge is added in the middle to connect the main channel with the two side channels. Mother cells are trapped underneath the pensile columns, while the smaller daughter cells are washed away by the flow. Please click here to view a larger version of this figure.
Figure 2: Fresh mother cells live longer than cells with unknown history. The replicative lifespan of fresh mother cells versus cells of unknown history (cells trapped from the beginning of the experiment) in SD media, 30 degrees. On average, fresh mother cells live about 2 generations longer. Please click here to view a larger version of this figure.
Figure 3: The length of budding intervals changes as cell aged. Budding intervals measured as time frames between buddings were color-coded, cells were ordered by their replication lifespan, and fresh mother cells were separated from cells of unknown history. Please click here to view a larger version of this figure.
Figure 4: Measuring the activity of Heat Shock Factor 1 (HSF1) using a microfluidic device. The Hsf1-activity reporter is constructed by a green fluorescent protein (GFP) fused to a crippled CYC1 promoter with HSE upstream13. Fluorescence images were taken every 30 min. GFP intensity was then quantified using a customized MATLAB code14. Data points for each single cell are connected and indicated with a colored line. In this experiment, the strain was grown in SD medium with 2% glucose (wt/vol) at 30 °C overnight; it was then diluted with the same medium for recovery. Afterward, SD medium with 0.05% glucose (wt/vol) was used to grow cells in the microfluidic chip during the fluorescence measurement. Please click here to view a larger version of this figure.
Movie 1: An example of fresh mother cells being trapped within the device for the whole lifespan. The movie shows a fresh mother cell born from a trapped cell. The red arrow at the beginning of the movie marks the position of the fresh mother cell and its first budding. This mother cell was trapped in the microfluidic device for its whole lifespan. Please click here to view this video. (Right-click to download.)
The PDMS device needs to be freshly made. Otherwise, the air bubbles caused by inserting tubes into the device will be difficult to remove. Step 3.4 is important to improve the cell loading efficiency by concentrating the cells. To increase the throughput of the experiment, 4 to 6 modules on the same PDMS chip connected to independently operating pumps are typically used to perform 4 to 6 different experiments (different strains or media compositions) simultaneously.
Compared to the conventional yeast replicative aging assay (which uses microdissection), the microfluidic method presented here is less laborious and time-consuming. Moreover, the microfluidic device allows for the detailed quantification of various cellular or molecular parameters, including cell size, cell cycle dynamics, cell morphology, and various molecular markers. This microfluidic method achieves long-term cell tracking with high-resolution microscopy by retaining mother cells under PDMS micro-pads as daughter cells are flushed automatically.
This device uses soft PDMS micro-pads to trap mother cells4, with the basic structure similar, as Huberts et al. described in his work7. There are a number of differences in the device design and experimental protocols. The wider PDMS pad in this device allows some newborn daughter cells to be trapped and analyzed (Figures 2 and 3, Movie 1). To identify such cells, we annotated the daughter cells budded from already trapped mother cells, ignoring the daughter cells that flushed away without budding. On average, we got about 2 fresh mother cells per PDMS pad. The ratio of these fresh mother cells to the cells of unknown history was about 1:2, among which about one third were kept in the device for the whole lifespan. These cells allow a more accurate lifespan measurement and make it possible to analyze correlations between mother and daughter cells. In this device, a deeper main channel is connected to two shallower side channels where the observations are made; this design helps to reduce the chance of the side channels being blocked by big air bubbles. For microfluidic chip production, this protocol also uses a simple method to bond the PDMS and cover glass, just using plasma exposure and oven baking, which increases the success rate.
With this protocol, the microfluidic device is able to robustly trap at least one cell (3-5 cells on average) per PDMS pad at the beginning of the experiment for wild-type haploid yeast strains (i.e., BY4741 or BY4742). About 30% of the cells can be retained throughout their whole lifespan. It is worth noting that the performance of the PDMS device depends on the gap size between the pensile columns and the glass and the size of the yeast cells. For wild-type haploid yeast strains, the suitable size for the gap is 3.5-4.5 µm. Outside this range, the loading efficiency and cell retention rate decline sharply. Thus, new devices for yeast strains with much larger or smaller cell sizes7 must be fabricated by modifying the height of the first layer made in step 1.
In summary, the device and protocol described in this article are not only suitable for yeast aging studies but are also applicable to other experiments that require the tracking of mother cells and the monitoring of molecular markers for many generations or throughout the lifespan.
The authors have nothing to disclose.
This research was supported by NIH Grant AG043080 and the National Natural Science Foundation of China (NSFC), No. 11434001. We thank Lucas Waldburger for proofreading the manuscript.
3'' <111> silicon wafer | Addison Engineering | ||
SU-8 2000 and 3000 Series | MicroChem | ||
SYLGARD® 184 SILICONE ELASTOMER KIT | ellsworth | 2065622 | Include Sylgard® silicone elastomer base and curing agent |
Petri dishes | VWR | 391-1502 | |
Harris Uni-core™ punch(I.D. 0.75 mm) | Sigma-Aldrich | 29002513 | |
24 mm x 40 mm SLIP-RITE® cover glass | Thermo Fisher Scientific | 102440 | |
3M Scotch Tape | ULINE | S-10223 | |
VWR® Razor Blades | VWR | 55411-050 | |
PURE ETHANOL, KOPTEC | VWR | 64-17-5 | |
WHOOSH-DUSTER™ | VWR | 16650-027 | |
5mL BD Syringe (Luer-Lock™Tip) | Becton, Dickinson and Company. | 309646 | |
PTFE Standard Wall Tubing (100ft, AWG Size:22, Nominal ID: 0.028) | COMPONENT SUPPLY COMPANY | SWTT-22 | |
Needle Assortment | COMPONENT SUPPLY COMPANY | NEKIT-1 | |
Desiccator | HACH | 2238300 | |
Lab Oven | FISHER SCIENTIFIC | 13246516GAQ | |
Nikon TE2000 microscope with 40x and 60x objective | Nikon | ||
Zeiss Axio Observer Z1 with 40x and 60x objective | Zeiss | ||
Syringe Pump | Longerpump | TS-1B |