Tissue Culture

Maintaining Sterility

The first and most important rule of tissue culture is that everything must remain sterile at all times. Make sure you fully understand how to maintain sterility while working. If your lab regularly cultures cells, you should have an appropriate tissue culture hood, and often a separate tissue culture room, that contains hoods and the cell incubators. This is because it is incredibly easy for cells to get contaminated, and once bacteria or other organisms settle into your cells, the culture is lost and you must start over. As you can imagine, if you have special lines or specific cell types that are hard to culture or obtain, this will set you back massively in your work. There are some basic rules to help you maintain sterility throughout each step of your work.

As you start out and get your reagents together for TC, remember to always wear clean gloves when you reach into the hood or into the incubators. Don't ever touch anything inside the hoods, incubators, or anything that is marked as sterile with your bare hands. Touching or opening things outside will contaminate them and make them unusable. Before putting your gloved hands into the hood or into the incubator, make sure that you spray them down with 70% ethanol to ensure sterility and cleanliness.

Anything and everything that goes in the hood must be sterile. That means that any reagents used inside the hood should NEVER be opened outside the hood. They should be purchased sterile (or sterilized in your lab by autoclaving). Before bringing them into the hood, they should be sprayed down with ethanol. Before taking them out of the hood, they should be tightly closed. They should be stored in a defined location where only sterile items are stored and there should be no mixing between sterile and non-sterile items. (Do pay attention to where they should be stored though - different items may need to be at 4 degrees, -20 degrees, or room temperature). The same applies to other items - pipettes, multi-channels, etc. - they should be delineated as sterile for the hood, be autoclaved, and then only opened inside the hood.

When you work in the hood, make sure that all your items remain sterile as you work. Do not ever open cells outside the hood. Cells go straight from the incubator to the hood and are only opened briefly inside the hood as needed to work. For reagents, maintaining sterility means that anything that touches the inside of your cells or reagent bottles must never touch your hands or any other surface in the hood. (For example, if you have a pipette tip that you are working with, make sure it never touches the outside of any bottles, your hands, or anything except the reagent it is meant for and the cells). Close or cap items as soon as you are done with them. In general, if something touches or if you are unsure, throw it out and use a fresh tip. Use fresh tips for every reagent and every cell type to avoid contaminating your reagents and your cells with other materials.

Once you are done with the hood, make sure to replace all the items, clean it well with 70% ethanol, and then close it and turn on the UV to fully sterilize it for the next person.

Recognizing Contamination

Given the discussion above, let's talk about how to recognize if your cells are contaminated. At first, it can be difficult to tell but you should check your cells every day for signs of contamination. Early signs of contamination include small black dots among your cells or low cell growth and increased detachment. As it progresses, the media will often change color and become progressively cloudy and eventually the cells will detach and die. As you develop experience, you may also notice that your incubator might start to smell a little funny if there is something in it that is contaminated.

A clean culture should look like this

Pulmonary Cell Culture - PromoCell
Note the clean cells, uniform shapes, and lack of black dots or oddly shaped cells.
There are also no floaters in the media that are visible
Image source: wikipedia

A contaminated culture may look more like this:

What is the reason for my cell-line contamination at day 3?
Note the floaters, the cell death, and the cloudy forms of bacteria covering the culture.
Image Source: ResearchGate
Maintenance & Fallbacks

Since the first rule is maintaining sterility and avoiding contamination, the next thing we talk about must be what to do if you do have contamination! The best thing you can do for yourself is to start out, if you are new to the process, with a cell line that passages easily, is relatively hardy, and one that your lab has plenty of stock for in case you do have contamination. Examples of these are 293T cells or HeLa cells, which most labs have in abundant quantities. It is also a good practice to separate your media and reagents from your labmates and to keep your cells in a specific part of the incubator so that you can isolate which reagents may be affected if there is contamination.

Once you are comfortable working with those cell lines and are confident that you can work without getting contamination, you can progress to more difficult cell lines. It is still always a good idea to keep your work materials separated from your lab mates, in case there are any issues.

If you do get contamination, the best thing to do is to start over. The contaminated plates should be soaked in bleach and then dumped. You should obtain fresh reagents that you are sure are sterile, clean out your incubator, sterilize it if possible, and then start afresh. If it is not possible to throw away the potentially contaminated reagents (expenses, etc.), another technique is to plate just the media overnight and see if it grows anything. If it does, you know it is the source of contamination, but if not, perhaps you can try again with the same reagents. If you truly are using a very precious cell line or resource, you may try treating with antibiotics, frequent media changes, and frequent washes to salvage the line. However, this is not a permanent solution if you have significant contamination.

To ensure that you have a fallback, anytime you obtain a cell line or create a line (with a knockout, transformation, or other modification), you should FIRST expand and freeze stock of the line. Freezing protocols are included below. Freezing the lines ensures that you will always have stock that you can start afresh with if you should need it. We would recommend freezing at least 5-10 vials if possible before proceeding with any major experiments.

Assessing Cells

Now that we've talked about avoiding contamination, let's briefly discuss the broader assessment and maintenance of your cell lines. The goal of tissue culture is maintain and propagate cells for experiments. If your lab depends heavily on cell models, chances are you will always have an incubator full of cells and at least an hour or two of your day will be spent maintaining those cells.

Part of that time, each day, should be spent looking at the cells and assessing for contamination and confluency, and then maintaining the cells appropriately based on what you find. The important things to do each day are the following

  • Check EVERY plate of cells and maintain only what you need for experiments and freezing
  • Identify cells that may be contaminated (if applicable)
  • Identify cells that are 80-100% confluent and require passaging
  • Identify cells that are not confluent but have not had media changes in 2-3 days
  • Identify cells that are required for experiments and need to be plated

The list above will determine your cell culture maintenance for the day. The contaminated cells should be disposed of to avoid spread to your other lines. Replate them from frozen if they are required for experiments. The confluent cells should be passaged out to new plates so they are less dense and have room to continue to grow and remain healthy. Cells that haven't had media changes should have fresh media to encourage growth. Finally, cells that are required for experiments should be plated out for those experiments assuming they are healthy and relatively confluent, along with additional maintenance plates so you don't lose the line. If the cells are not ready enough that you could plate your experiment and maintain extra cells, they should be given a few more days to continue growing.

Major TC Steps

In this last section, I want to briefly discuss how I think about the steps involved in TC. To me, there are four major steps:

  1. Assess cells and decide your plan
  2. Detach cells from the current plate
  3. Separate the cells from the media
  4. Isolate the cells and re-plate them for an appropriate application (maintenance vs experiment)

We discussed the first step above. This is key and easy to get lazy about but it is essential to make sure you have a good stock of cells at all times to be able to produce data.

The second step typically involves washing your cells, removing the media and then adding in some sort of enzymatic detachment solution (trypsin, EDTA, etc.). This will detach the cells from the plate and allow you to remove them so that they can be transferred to a tube.

The second step, once the cells are detached, is to separate them from the old media. This is usually done with centrifugation in 15mL or 50mL tubes. Since the cells are heavier than the media, they will settle to the bottom when spun. This will allow you to then remove the old media and maintain the cell pellet. It is important here to not lose your pellet when removing the media.

Once the pellet is isolated, you can do anything you want! Step 4 is our decision point. We can either decide to freeze the cells here, using a special freezing media. Or we can plate the cells for an experiment (with additional maintenance plates) which will require counting and then plating cells in appropriate dishes dedicated to our experiment. Or we can simply re-plate the cells into fresh plates at a lower density. When you do this, remember that you do not have to re-plate all the cells - just plate what you think you will need and either freeze or dispose of the rest.

These steps are discussed in more detail in the protocols below.