Measuring the replicative lifespan of Saccharomyces cerevisiae using the HYAA microfluidic platform

Abstract

The replicative aging of the budding yeast, Saccharomyces cerevisiae, has been a useful model for dissecting the molecular mechanisms of the aging process. Traditionally, the replicative lifespan (RLS) is measured by manually dissecting mother cells from daughter cells, which is a very tedious process. Since 2012, several microfluidic systems have been developed to automate the dissection process, significantly accelerating RLS determination. Here, we describe a detailed protocol of RLS measurement using a commercially available microfluidic system based on the HYAA chip design, which enables data collection of up to 8,000 cells in a single experiment.

1. Introduction

The budding yeast, Saccharomyces cerevisiae, is one of the most genetically tractable eukaryotic models in aging studies. Yeast cells undergo asymmetric cell division, producing a smaller daughter cell from a larger mother cell. The smaller daughter cell can be physically dissected away from mother cell, hence the cell division events for a particular mother cell can be counted. A yeast cell can only produce a limited number of daughter cells, that is a finite replicative lifespan (RLS), before entering a permanently arrested state followed by cell lysis. Studies in yeast replicative aging have led to the discovery of several conserved molecular pathways that regulate aging in diverse eukaryotic species.

Traditionally, RLS is measured by physically separating daughter cells from mother cells using fiber-optic needles under microscope (1), by which a round of RLS measurement typically takes 4-6 weeks. An experienced dissector usually handles no more than 300 cells in an experiment, making this procedure very labor-intensive and time-consuming.

Since 2012, several microfluidic designs have been developed to enable fast and reliable measurement yeast RLS. In all these systems, yeast cells are fixed in microfluidic channels via different mechanisms and daughter-mother cell separation is driven by medium flow. When coupled with time-lapse imaging, cell division events for the entire replicative lifespan of hundreds of single yeast cells can be tracked, recorded, and counted. These methods have drastically reduced the time required for RLS measurement and increased experimental throughput. Specifically, the high-throughput yeast aging analysis (HYAA)-chip developed by our group enables simultaneous RLS measurement for 16 different strains, generating data containing up to 520 cells per strain or 8,000 cells in total, in a short period of 72 hours. The HYAA-chip design has been used in several published yeast aging studies (2-5) and proved to be robust and consistent.

2. Materials, Reagents and Equipment

2.1. Sample preparation

1. Yeast Extract–Peptone–Dextrose (YPD) Medium: 1% Yeast extract, 2% Peptone, 2% Dextrose, filter sterilized.

2. Yeast strains.

2.2. Setting up microfluidic experiment

1. 50ml syringes (Air-Tite™ HSW Soft-Ject™ Luer Lock Disposable Syringe)

2. Luer-stub adapters (BD Intramedic™ PE Tubing Adapters)

3. Non-DEHP medical grade tubing (Saint-Gobain Tygon™ ND 100-80 Tubing)

4. Computer-controlled dual syringe pump (Innovative Biochips)

5. HYAA microfluidic chip (Innovative Biochips)

6. Microscope (fluorescence) with autofocus, time-lapse abilities and preferably an automated X-Y control stage (e.g., A1 Cell Imaging System; Innovative Biochips)

7. Hollow pins (Type 304 stainless steel pins; Innovative Biochips)

8. Solid pins (Type 316 stainless steel pins; Innovative Biochips)

2.3. Loading yeast cells into HYAA-Chip

1. 1ml syringes (BD Luer Lock Disposable Syringes)

2. Computer-controlled multi-syringe pump (Innovative Biochips)

2.4. Time-lapse imaging

1. Microscope temperature control system (e.g., Onstage Incubator; Innovative Biochips)

3. Methods

All procedures are to be performed under room temperature. A typical a microfluidic system is depicted in Figure 1.

Figure 1:

Microfluidic platform for yeast RLS measurement. Up: The platform consists of pump system and live cell imaging system connected with environmental chamber control unit.

3.1. Sample preparation

1. Culture yeast cells overnight (see Note 1). On next morning, transfer 50μl of cultured cells to 1ml of filtered YPD medium (see Note 2). Keep on ice until experiment.

3.2. Setting up microfluidic experiments

1. Fill 50ml syringe with liquid YPD medium or other medium of choice (see Note 3). Dispose of any air bubbles in the syringe. Connect syringe with luer-stub adapters and non-DEHP medical grade tubing with hollow pin, then fix onto a computer-controlled dual syringe pump for supplying media. Turn on the media supply syringe pump and make sure liquid flow is not obstructed (see Note 4). The flow rate is set at 20μl/min (see Note 5).

2. Connect syringe with the medium inlet of HYAA-Chip once medium flow is stable (see Note 6). Wait until medium comes out from cell loading inlets (Figure 2), then insert solid pin (see Note 7) into cell loading inlets to block the flow. Connect tubing to outlets for disposal (see Note 8).

3. Check the HYAA-Chip under microscope and wait until all channels are filled with medium.

Figure 2:

Schematic design of HYAA microfluidic chip.

3.3. Loading yeast cells into HYAA-Chip

1. Thoroughly vortex diluted yeast media prepared in A and fill 1ml syringe (see Note 9). Dispose of any air bubbles and connect with luer-stub adapters and non-DEHP medical grade tubing. Install syringe on the syringe pump for loading cells, turn on the cells loading syringe pump and wait for medium flow to stabilize. Multiple syringes can be installed at same time if using a computer-controlled multi-syringe pump.

2. Remove solid pin blocking loading inlets, connect syringe with loading inlet on HYAA-chip, set initial flow rate of the cells loading syringe pump at 1μl/min (see Note 10).

3. Closely monitor channel loading status under microscope to ensure no cross-contamination is happening (see Note 11).

4. When cells are loaded at desired rate, remove connected tubing and insert solid pin back to stop the flow (see Note 12).

5. Turn off the cells loading syringe pump.

6. Set the medium flow rate of the media supply syringe pump to 10μl/min.

3.4. Time-lapse imaging

1. Set up beacons for imaging, ensure that at least 100 cells are captured. The magnitudes should not be less than 20x.

2. After setting up beacons, turn on automatic focusing, set chamber temperature to 30°C and humidity to off. Set experimental time as desired.

3. For determination of replicative lifespan, image cells every 10 min. When using fluorescence microscopy, it is preferred to image the cells less frequently (e.g. every 20 min) to avoid phototoxic effects. It is recommended to regularly check the focus of the images during the experiment and, if necessary, adjust it.