シングル出芽酵母細胞での寿命と細胞表現型を測定するためのマイクロ流体デバイスを使用しました

The overall goal of this experiment is to make a specialized microfluidic device and use it to measure the lifespan and cellular phenotypes of a single yeast cell. This method can help answer some of the key questions in the yeast aging field, such as why mother cells age and die and what are the earlier molecular events that drive the loss of cellular homeostasis and eventually cell aging and death? The major advantage of this technique is that we can track mother cells, track their lifespan, and the various molecular markers at the same time, so we can look for molecular events that correlate with lifespan.

Though this method can provide insight into yeast aging it can also be applied to other areas, such as monitoring secondary responses to participation that requires cell checking for many generations. Generally, individuals new to this method will struggle because microfluidic device fabrication, cell loading, microscope set up require a lot of practice to perform successfully. Prior demonstration of this method is critical as it is difficult to properly load the microfluidic device with yeast.

To begin fabricate a silicon wafer mold for the device using standard techniques as described in the accompanying text protocol. Place it feature side up into a 15 centimeter Petri dish. Secure the mold on the Petri dish with tapes.

Next place a clean weighing boat on a balance and tear the balance. Pour 50 grams of PDMS base into the weigh boat and then add five grams of PDMS curing agent. Stir the PDMS base and curing agent with a disposable pipette.

Start from the edge of the weigh boat and slowly move inwards. Stir the mixture thoroughly for several minutes until small bubbles form throughout the mixture. Pour the mixture slowly into the Petri dish, so it completely covers the silicon wafer mold.

Then place the Petri dish in a vacuum for 10 minutes to remove all air bubbles from the PDMS mixture. If bubbles remain on the surface of the mixture once removed from the vacuum use a pipette to blow them out. Next, bake the device at 75 degrees Celsius for two hours.

When cooled back to room temperature, carefully cut through the PDMS around the mold and then gentle peel the layer of polymerized PDMS off the silicon wafer mold, avoiding any damage to the construction of the wafer mold. Place the PDMS layer on the bench with the pattern side facing up. Carefully cut out the individual chip with a single edge industrial razor blade, retaining an adequate margin from the edge of each single chip to avoid construction damage.

With the pattern side facing up, use a punch pen to punch holes straight down through the inlet and outlet circles on each side of the channels. Check each hole by inserting the punch pen needle again into the hole. Make sure the needle can come out from the other side, indicating that there are no blockages.

Next use the tape to clean dust particles and debris from the top surface. Repeat this step at least three times, and leave a clean piece of Scotch tape on the PDMS to maintain a dust free surface. Then turn the device over and repeat this procedure on the opposite site of the PDMS.

Leave the last piece of tape on this side as well. Now prepare a glass surface for the base of the device. Select the 24 millimeter by 30 millimeter piece of cover glass with a thickness between 13 and 17 millimeters.

Spray 70%ethanol on the glass, and dry it with dust remover to sterilize the surface. Then transfer the cover glass and PDMS to a plastic plate. Remove the Scotch tape from the PDMS, and place the pattern side facing up in the plate, without contacting the top surface.

Then transfer the plate into a plasma chamber. Apply an oxygen plasma treatment to the PDMS and the cover glass to render the surfaces hydrophilic. Then remove the plate from the plasma chamber and carefully connect the plasma treated surfaces of the PDMS in cover glass.

Ensure that there are no air bubbles between the PDMS and the cover glass. Finally, transfer the device to an oven and bake at 75 degrees Celsius for at least two hours. Submerge the input and output tubes in 70%ethanol solution one day before PDMS chip preparation.

The next day fill a five milliliter syringe with autoclaved water, and wash each tube by placing the tube on the syringe and flushing the tube with water. Repeat the washing step at least three times to remove residual ethanol. Next examine the PDMS channels under an optical microscope.

Using a 10 X objective make sure that the structures are consistent and intact. Stabilize the intact PDMS chip on the microscope platform using Scotch tape. Secure a five milliliter syringe loaded with sterile water to a syringe pump and insert the corresponding input and output tubes to the device.

Set the flow rate to 750 microliters per hour and rinse the chip for about 10 minutes. Watch the channels to ensure that any air bubbles are washed away. If any bubbles remain following the 10 minute rinse manually adjust the speed to eliminate the bubbles.

Transfer one milliliter of a prepared yeast sample into two 1.5 milliliter microcentrifuge tubes and centrifuge the samples for five minutes at 3, 000 times G.Remove the majority of the supernatant from each tube and combine the remainder to form a 5 milliliter sample. With the yeast sample now ready, remove the input tubes from the PDMS chip. Manually reverse the syringe pump to suck in a small air bubble that is one to two centimeters in length, each input tube, and then suck in cell cultures.

Then insert the input tube back into the PDMS chip and restart the pump to load the cells at a speed of 750 microliters per hour. Leave the syringe pump on for 10 minutes and then examine the cell loading progression under the microscope. A successful loading will trap cells under more than 60%of suspend pillar.

To maintain the yeast cells, switch the solution in the syringe to a nutrition solution, and adjust the speed to 400 microliters per hour. The lifespan phenotype of yeast can be obtained by counting the number of buds produced by each trapped mother cell, and estimating the Kaplan-Meier estimator. Using this protocol the device frequently traps fresh daughter cells that budded off from the already trapped cells.

These cells then turn into fresh mother cells which can be identified using downstream image analysis software. These fresh mother cells provide a more accurate lifespan measurement. In this experiment, the average lifespan of the fresh mother cells was slightly longer by about two generations.

In addition to the number of buds, the time interval between two successive budding events can be extracted from the imaging data. Budding slows down dramatically towards the end of their lifespan indicating the unhealthy state of these aged cells. While attempting this procedure, it's important to remember to both use a freshly made chip to help avoid bubbles, and to increase the cell concentration in order to improve cell loading efficiency when needed.

After its development this technique paved the way for researchers in yeast aging field to explore the early molecular changes that are causal to the aging phenotype. After watching this video you should have a good understanding of how to make this microfluidic device and how to use it to measure lifespan and cellular phenotypes in single cells.