# Cell Culture 101  1

https://www.youtube.com/watch?v=dSxH9xNmhWg

[00:02] Hello and welcome.
[00:03] My name is Laura.
[00:04] I am a molecular researcher here at the University of Toronto.
[00:08] Today we will introduce you to the basics of cell culture.
[00:11] Cell culture is the growth of eukaryotic cells in vitro for scientific purposes.
[00:17] Today we are using the MCF7 breast cancer cell line to grow up, culture, harvest, and treat so that you can see them for your lab purposes.
[00:29] One of the most important things about working in cell culture is that we maintain a sterile working environment.
[00:34] In order to achieve this, we use airflow hoods like this.
[00:39] Airflow hoods provide a barrier against the external environment because if things like mold or bacteria were to get into your samples, they would invalidate the results and you would need to start again.
[00:49] Airflow hoods work by generating a barrier between the external environment and the internal working environment.
[00:56] How they do this is there is a current of air that is pulled down into the flow hood and then back through a system of.
[01:04] Filters so that the air in the hood itself is sterile.
[01:06] What we will be doing is we also use ultraviolet lights that are mounted on the back of the hood.
[01:14] And those lights irradiate the working surface so that there is no potential for contamination.
[01:19] The first and most essential reagent that we will be using is cell culture media.
[01:23] In this case, we're using DMEM or Dulbecco's Modified Eagle Medium.
[01:27] This media provides salt balance, pH balance, and essential nutrients so that the cells can survive.
[01:32] We also supplement this media with 10% fetal bovine serum.
[01:37] Fetal bovine serum is the plasma from a fetal cow.
[01:40] And what we do is we harvest that because it contains a large number of growth factors that will then stimulate our breast cancer cell lines to grow.
[01:49] We also use a 1% prophylactic administration of penicillin and streptomycin.
[01:55] What that does is it prevents any potential bacterial contamination that may occur.
[02:01] In order to prep this bottle, um, we.
[02:05] Spray it using a 75% ethanol solution that disinfects the surface of the bottle, and it is now ready to be placed in the cell culture hood.
[02:18] The next reagent we are using is one times phosphate buffered saline.
[02:24] This is just a basic saline that is buffered to pH 7.3 that we can use to wash off any lingering remnants of media that may be on the cells.
[02:31] Again, it needs to be disinfected.
[02:41] Finally, the last reagent is one times trypsin EDTA.
[02:44] This is a digestive enzyme that digests the external cellular proteins so that the cells lift up from the bottom of the plate and are in suspension.
[02:54] From that, we can then dilute them and replate them in different plates.
[02:59] That is called passaging.
[03:08] Cells now that our reagents are warmed and disinfected and we've placed them in the cell culture hood, we are ready to begin the process of cell culture.
[03:17] The MCF7 breast cancer cell lines are adherent cells, meaning that they adhere to the bottom of the culture vessel that they are being grown in.
[03:23] As you can see, there are multiple examples of culture vessels.
[03:28] This one here is a cell culture dish; the lid comes off, and there are multiple sizes that can be used depending on the nature of your experiment.
[03:39] This is a cell culture flask.
[03:42] Cell culture flasks, as you can see, are fully sealed and they have a lid.
[03:47] There are pros and cons to both the dish and the flask.
[03:50] Dishes are easier to work with, but there's a higher potential for contamination as the lid comes right off.
[03:57] Flasks are a little more awkward to work with, but they are protective of the cells because they have a lid.
[04:04] For the purposes of this video, we will be using the cell culture dish.
[04:08] This is a cell culture incubator.
[04:11] Maintains conditions of 37° C and 5% CO2 which are similar to the internal conditions of our bodies.
[04:17] Our cells are growing in a cell culture dish with inside.
[04:21] Let's take a look.
[04:29] As you can see, there are many cell types being cultured in this incubator.
[04:31] Our cells are the MCF7 cells, and now that we have them from the incubator, we can take them and passage them in the flow culture hood.
[04:41] Now we are ready to passage our MCF7 cells for our experiment.
[04:43] How we do this is we will remove the existing media, rinse the cells with PBS, and then place trypsin on the cells.
[04:51] The trypsin will digest the external proteins of the cells, allowing them to lift up off the bottom of the dish.
[04:58] Then we will dilute the cells in new media and place the newly diluted cells into new plates that contain new media so that the cells can now grow in different size plates for our experiment.
[05:39] [Music]
[07:15] is
[08:12] [Music]
[08:21] Initially, after the cells have been passaged, they will be quite separated and will have a lot of space around them.
[08:26] The population density of cells in your plate is called cell confluence.
[08:30] Confluence is measured subjectively by comparing the amount of space surrounding the cells.
[08:34] If the cells cover 30% of the plate, then the plate is 30% confluent.
[08:40] If there is virtually no space around the cells, then the plate is 100% confluent.
[08:45] It is not a good idea to let cells reach 100% confluence.
[08:47] Generally, when cells are too close together, a phenomenon called contact inhibition comes into play.
[08:53] Apoptotic pathways are then upregulated in the cells, and they begin to die.
[09:01] In order to prevent this, we passage the cells before they reach 100% confluence.
[09:05] Now that we've passaged our cells, we put them back into the incubator and allow them to adhere back to the bottom of the plate.
[09:13] Once the cells have adhered, then we can treat them with our estradiol and/or vehicle, and that's what we will be doing next.
[09:18] So in.
[09:21] In order to treat them, we have previously calculated dilutions of ethanol or its vehicle aliquoted in media.
[09:29] These aliquots then get put into the plates and allowed to grow for the appropriate time limits: 12, 24, and 48 hours.
[10:04] [Music]