Patterning di cellule staminali embrionali che utilizzano il chip Bio flip

I am nickel Mital. I'm a graduate student in the Bowman lab. Today I'm gonna talk about device developed by two former students in our lab, which is called the bio flip chip.

And this device enables the delivery of cells on a substrate of the experimenter's choice in a configuration of the experimenter choice. So the device itself, we have one of them here is very simple. It basically consists of tiny wells in a piece of silicone.

Each well is usually designed to just hold a single cell, but if required, we can of course make them larger. The fabrication of the device is made so that the wells are in the same arrangement that cells will eventually end up in on the substrate. So the device itself is made using soft lith photographies.

In particular, we use PDMS, which is poly dimethyl suboxane, and mold that off of a silicon wafer. We have one of them here. So the mold wafer itself is made using standard photolithography to pattern an SUA photo resist, which is spun onto the silicon wafer.

So in a nutshell, cells are transferred onto the substrate of choice by basically filling the wells up on the bio flip chip with these cells. We then apply the chip to the substrate of choice, often a tissue culture of polystyrene. And then finally, we flip the combination over allowing cells to fall out of the wells and onto the substrate.

I'm Stephanie Flavin. I'm an undergraduate researcher here in the Goldman lab, and today I'll be demonstrating how to use the bio flip chip to pattern cells the day before. We wanna do a patterning experiment.

We'll plasma bond the PDMS to a one inch by one inch glass slide. And then we soak the chip overnight, well side down and a PBS solution. We'll also cut gaskets to be used in the patterning experiment and collect binder clips to be used as well.

Apply 200 microliters of BSA to the surface of each chip. Agitate the surface of the chip of the pipette tip to get out bubbles and allow BSA solution to fill wells. Let the chips sit under the hood for one hour in UV light passage cells as normal.

After one hour aspirate the BSA solution off of the chips and wrench each chip with 200 microliters of PBS apply 200 microliters of passage cell solution at a density of 10 to the six cells per mil. After five to 10 minutes of waiting for the cells to fall into the wells, rinse the surface of the chip with PBS. Tilt the chip to one corner at approximately a 15 degree angle and slowly pipet off the cell solution from the bottom corner of the chip.

Return the chip to level and slowly add 50 microliters of PBS to the upper corner. Repeat the washing step as many times as necessary. Once xls have been washed from the surface of the chip, add approximately 50 microliters of media to the inner gasket of the dish.

Add media to the top of each chip. Next, invert the dish and tilt and lower onto the surface of the chip, starting at one corner. And then flattening the chip on, flattening the dish onto the chip so as to avoid the formation of bubbles between the dish and the chip.

Add binder clips to each side of the chip to secure it into place. And remove the handles from each binder clip so that when the chip is flipped over, it will sit level on the dish, move the chip to the incubator and flip quickly, avoiding and avoiding all unnecessary movement.