Transitioning from prokaryotic cells to eukaryotic cells is a big step-up from every other lab done up until this point. Not only does it require more caution to remain aseptic, but it also requires constant monitoring over a much longer period of time. A single infect or a small slip in the maintenance schedule could mean the death of your entire culture (aka weeks/months of work wasted). While it may be daunting at first, it doesn't take long to get acclimated.
The cell line I worked with in my class was the Sf9 line, cells that ordinated from the ovarian cells of Fall Armyworms (Spodoptera frugiperda). Compared to many other types of cells, Sf9 cells grow rapidly and under much easier conditions (standard atmosphere and room temperature conditions). This eliminates the need for equipment such as incubators, humidity control, CO2 control, and more. Despite the line used, there techniques remain relatively constant. The two most important techniques for first-timers that will be discussed are feeding and subculturing.
Review of Aseptic Technique
Before going into cell maintenance, it's important to understand aseptic technique. Aseptic technique aims to keep both you and the sample you're working with safe. While labs intended for education at lower levels have lots of leeway for aseptic error, future cultures can be extremely delicate or pathogenic, requiring deliberate movement to ensure everything stays in their place. For labs taught at a typical high school level, aseptic technique consists of proper PPE, sterilizing tools, washing hands before/after a lab, and never putting things down. To handle eukaryotic cells, basic procedures are expanded upon to make the environment even cleaner.
The first big addition is sterile handwashing. Commonly used by surgeons before operating, sterile hand washing is intended to remove all microorganisms and debris from your hands before donning gloves. To sterilize your hands, take off all jewelry, and wash your hands all the way to the bottom of your elbow using soap. Make sure to wash every part of your hands (even the bottoms of your fingernails) by using the chart below. Then dry your hands and turn off the sink with clean paper towels, making sure your hands don't come into contact with anything in the process.
After handwashing and donning the rest of your PPE*, you will clean your gloves with 70% ethanol or a similar alcohol. This is assuming that what you've donned are non-sterile gloves. If you have sterile gloves, this step isn't necessary. However, the price tag (and availability if you're living in a pandemic) of sterile gloves make it unreasonable for most educational labs. Ethanol allows us to sterilize our gloves before and during the lab and eliminates the need to grab new sterile gloves every time a possible contamination occurs, something that happens frequently for students.
The final big addition to our aseptic toolkit is the sterilization of practically everything. Bottles, flasks, tables, microscopes, you name it. Everything must be cleaned with 10% bleach or 70% ethanol as to make sure your culture never gets infected with anything. When using a culture flask, for example, the lip and the outside of the bottle would be cleaned thoroughly. This may seem to be the simplest of the new additions, but an element of speed is crucial. Once again assuming you don't have a clean room or a laminar flow hood, the air around your class lab is filled with microbes. These microbes may fall onto your equipment and try to kill your culture. To make aseptic technique a little easier, a Bunsen Burner may also be used to create a 30 cm radius that'll remain sterile for as long as the fire is active, something that is more common when repeatedly opening a culture container for longer periods of time.
*PPE for this lab consists only of gloves, goggles and a surgical mask, but may also include an isolation gown.
Culture Maintenance: Feeding
Now onto the first of two essential cell line maintenance skills: culture feeding. Like with all cells, nutrients is required to grow healthy and happy cultures. However, eukaryotic cell media differs from prokaryotic cell media in that the media eukaryotic cells use contains many more types of macromolecules such as amino acids, glucose, serums, growth factors, and attachment factors. These molecules are used much more rapidly than prokaryotic cells with their nutrients, yet eukaryotic cells grow at a much slower rate. This means that simply inoculating new media with cells isn't a viable option if you want to keep a cell line alive.
Feeding solves this issue by replenishing the media instead of replacing it entirely. To feed a cell culture, you simply discard X** mL of old media and add X** mL of new media. After that, the culture is placed back into incubation. Once in incubation, the procedure is completed. When discarding old media, effort is put in to take media from the top of the container rather than the bottom. This is because all the cells, if not in suspension, are attached to the side of the container. This means that collecting media from the bottom of the container runs the risk of discarding perfectly healthy cells.
**Different cell lines and different protocols call for different amounts of media to be discarded. In most cases, however, feeding results in the same volume of media to be in the container before and after.
Culture Maintenance: Subculture
The second essential cell line maintenance skill that'll be covered is subculturing. Before that, the concept of confluency must be learned. In simple terms, confluency is the percentage of the surface of a culture flask that is covered by cells adhered to it. So a culture at 50% confluency covers half of a culture flask with adherent cells. Confluency is one of the many indicators used to be able to predict the current stage of cell growth a culture is at or when maintenance must be done. For example, Sf9 cells at 80% confluency are in the stationary phase. Now that confluency is understood, subculturing can be explained easily.
Subculturing is when cells are taken from an existent culture and placed into new media, creating a new culture. This is used when preparing an experiment on a culture, preserving cells for later use, or when creating a new family of cells with different genetics. The concept is quite similar to the inoculation of bacterial media, but takes a couple more steps. The culture to be subcultured is first knocked to put the cells into suspension, which is then verified with a microscope. The cell media is then added into a new container with new media and rocked to spread the suspended cells. The new container is checked to see whether there are viable cells and no contamination before placed into incubation. Once in incubation, the procedure is completed. Before subculturing, it's important to check the confluency to make sure there's an ample number of cells to transfer over. The exact percentage may very for different cell lines, but it typically ranges between 80% to 100%.
Eukaryotic cell research is truly the cornerstone of modern biological research. Allowing us to be able to do research on cells similar to ours enables us to develop drugs or treatments that can be used on a variety of conditions. There are many more advanced techniques for such purposes, but every successful cell biologist can say they've mastered feeding and subculturing before they've got to where they are. And as with many skills, mastery takes time and patience. The most common error is contamination from viruses, bacteria, and more. I recommend taking time to first master aseptic technique and steading your hands when transferring media. After many attempts, it becomes as easy as pie. Sooner or later, you can brag to all your nerdy friends how cool and skillful you are!