Debbie Knight

A Day in the Life: March 23, 2012

In research log on March 23, 2012 at 9:00 am

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab on a particular day. These entries I will call “A Day in the Life…”

A couple of days ago, I wrote about how to start up a cell culture — from the deep freeze. Today, I will discuss how to move the cells from one culture flask to a larger one (or ones). This process is called “passing cells” or “passaging cells.” And it’s something you do if you need lots cells for an experiment or experiments.

I’m working with cells that were isolated from tiny blood vessels in the human brain — they’re officially called “human brain cortex microvascular endothelial cells” but in our lab we call them BMVEC (we pronounce it”buh-muh-veck”).

The cells were brought out of the deep freeze (or cryopreservation) three days ago and they have divided and grown. Here’s how they looked after the first 24 hours:

The cells 24 hours after passing. They are now completely attached and have gone through a round or two of cell division. Time = 24 hours

And after 72 hours…

Cells after three days in culture. They have gone through many cell divisions and have filled in all the empty spaces. We call this a "confluent monolayer" and these cells will be moved from this flask to a larger one.

The culture medium is removed. The cells, still attached to the growing surface, are washed twice with phosphate-buffered saline. This is to remove any residual medium and unattached (presumably dead) cells from the flask. You need to do this before added an enzyme that will detach the cells from the growing surface — there are components in the culture medium that will neutralize the enzyme’s activity.

A small amount of an enzyme called trypsin is added. This enzyme chews on certain amino acids in proteins — and the cells are attached to the bottom of the culture flask by a thin protein layer. So the idea is to treat the cells long enough that the enzyme only chews on those proteins and not on the cellular membranes. So you need to monitor the cells. The enzyme can detach the cells in a few seconds to a couple of minutes, depending on how long the cells have been in the culture flask (many cells can make their own attachment proteins, so the longer they’re in the flask, the more protein, the tougher it is to get them off the flask’s surface) and the strength of the enzyme solution.

Here is a series of photos showing the cells detaching from the flask’s surface.

Moments after trypsin (an enzyme) is added, the cultured cells begin to round up. This is seen under the microscope as they are going from a dark color to a white color.

About 30 seconds later, more of the cells are rounding up as they detach from the culture flask's surface.

Here all the cells have rounded up. At this point a sharp tap on the bench top will dislodge the cells completely from the culture flask's surface.

Once the cells have been detached from the surface of the culture flask, they are still in the enzymatic solution. The trypsin needs to be neutralized so that it will not damage the cell membranes, essentially killing the cells. To do this, culture medium that contains blood serum is added. The serum acts as a enzyme target as well as contains factors that inactivate the enzyme.

The cells have been detached from the growing surface of this culture flask using an enzyme called trypsin. After detachment, the enzyme was neutralized by adding culture medium.

The cells are then pelleted out of this solution using a centrifuge. And once pelleted to the bottom of the tube, the solution is gently removed. Culture medium is added to the pellet, the cells are mixed and transferred to new culture flasks.

In my case, the cells had been growing in a culture flask called a T75 which has a growth surface area of 75 square centimeters. For the BMVEC, they need buddies around to grow, so you can’t pass them too thin into another culture flask. They can should be passed at about a one-to-four ratio. This means that if they were growing in ONE T75, I can only pass them into FOUR T75s or an area approximately equivalent to those four T75s (i.e., 300 square centimeters). In my case, I am passing them into two T162 flasks — slightly more surface area than I just mentioned (324 square centimeters to be exact), but because it’s the next sized flask we have in the lab, I’ll be using them.

The cells are in the top flask and will be passed into two larger flasks so they continue to grow.

Here the cells have been moved (or passed) from the small flask on the right to the two larger flasks on the right.

Flasks are placed in the incubator so the cells will grow to cover the bottom surface of the culture flasks.

And there you have it, Passing Cells 101.

For the experiment I need to do, I will need quite a few more of these large flasks, so I will be repeating this process a couple of times — each time increasing the number of culture flasks I am working with. Next passage will give me eight flasks. And the following passage will give me 32 flasks.

How often do I pass the cells? It generally depends on how “happy” the cells are as they grow, but usually three to five days. So hopefully in a couple of weeks, I’ll be able to do my experiment.


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