Everyday Lab Experiments - Part 1: Cell Culturing

When studies are reported, it is most often the results that are focused on. Extraordinarily little attention is paid to what experiments were used to attain the ground-breaking results. In medical research, there are many experiments that are routinely used to explore research questions. This multi-part series will explore a few that are quite commonly used regardless of area of medical research.

First, most medical research involves using living material, ideally as close as possible to what a doctor would see in a clinic. This is where cell culturing comes in, which is a technique that involves growing cells in a controlled environment outside the body. Cells are a crucial material to understand how a disease might progress, how a treatment may work, or just to understand how cells behave in certain conditions. Cells are the living unit that make up our entire body, and when they stop working properly, it affects our health. Being able to study them in an isolated environment where they can be manipulated to mimic diseased environments is a crucial part of medical research.

As fun as it would be to have cells listen to music, watch plays, and read novels, cell culturing merely involves maintaining and growing cells, albeit outside a living entity. Medical research focused on understanding disease involves using living material, ideally as close as possible to what a doctor would see in a patient. Although many science experiments would benefit from having access to human tissue donated by patients, it is not always readily available. Additionally, it takes more time and resources to submit and then receive ethics approval to procure donated tissue from patients. Sometimes proof of concept is required before permission in working with human subjects is granted, and testing a hypothesis using cells is a good first step in gathering important data towards a more comprehensive study. This is where cell culturing comes in, which is a technique that involves growing cells in a controlled environment outside the body. Cells are a crucial material used to understand how a disease might progress, how a treatment may work, or just to understand how cells behave in certain conditions. Cells are the living unit that make up our entire body, and when they stop working properly, it affects our health. Being able to study them in an isolated environment where they can be manipulated to mimic diseased environments is a crucial component of medical research. Scientists can acquires hundreds of different types of cells from life sciences companies. These humanely acquired cells are then grown in an environment that mimics the human body. Different cell types sometimes require different parameters, but the majority of cells are grown on plastic culture dishes in incubators that maintain a 37˚C temperature and 20% oxygen concentration ¹ . In fact, even cells collected directly from patients can also be cultured.

Inducible pluripotent stem cell (iPSC) sitting on feeder cells (fibroblasts)

One of the primary goals of cell culture is to increase the number of cells. They are very, very small, with cells in our body ranging from 10-100 µm in diameter, which is smaller than the width of human hair. Because of their small size, often millions of cells are needed to be able to measure significant changes that may have occurred within cells during experimentation. Cell culturing is an excellent way of growing large numbers of cells. Not only is it important to grow lots of cells, it’s very important to grow the intended type of cell. Currently, the majority of cell culturing is mono-culturing, where a single type of cell is grown in a single layer at the bottom of the culture dish. When performing experiments, it is important to remove as many variables as possible, and cell culturing allows for the controlled expansion of specific types of cells. There are also examples of cell culture with more than one type of cell. For example, inducible pluripotent stem cells (iPSCs) (which I discuss here in this article discussing stem cells ) require a layer of feeder cells, fibroblasts, in order to grow and maintain their identity as stem cells. These fibroblast cells essentially are important to support the growth of iPSCs, kind of like providing a good home environment for a growing family. Cell culturing isn’t always flat either, although this is still the most common method of increasing cell numbers. Within the tissue engineering field, there significant innovation of traditional cell culture techniques to move towards 3-dimensional cell culturing. This is because, in our bodies, our tissue exists in 3-dimensions and medical research strives towards using physiologically relevant experimental models to best mimic what is happening in the body. The closer an experiment is able to accurately replicate what is happening within a body, the greater its accuracy, whether it be when treating disease or understanding how diseases progress. Having 3-D culture systems, where cells can grow into tissues as oppose to a single flat layer, is significantly more representative of what happens physiologically.

The basics of how cell culture works are fairly straight-forward, although the equipment required to store, and culture cells is less so. Cells are delivered to the lab in frozen vials, and then kept in liquid nitrogen containers which are maintained at ~-196˚C. When ready to be used, they are thawed by being placed in a water/dry bath at 37˚C. Everything being used to maintain cells throughout the culture process are also required to be at 37˚C to prevent cold damage to the cells. Covering cells with clothes and blankets is highly impractical (and would also introduce contaminants) but having them hanging out and growing in a sauna-like environment is a perfect way to maintain them.

In fact, the incubators where cells are stored are essentially large cell saunas, with temperatures maintained at 37˚C, a constant supply of 95% O2 and 5% CO2, and pool of water at the bottom of the incubator to maintain humidity. Sounds pretty cozy, eh?

Just like us, cells need nutrients to live. In fact, the nutrients that we eat are what cells need in order to thrive. To feed cells in a culturing environment, a liquid nutrient supplement (food) called “media” is used. Although this term may seem like a lexicological nod to cell “culture”-ing, this media is merely referring to plural term for “medium”. Cell culture medium contains vitamins, inorganic salts, amino acids, glucose, growth factors, hormones, and attachment factors that are required by cells to thrive and multiply². Any of those sound familiar? Cells do not have mouths with which to ingest their nutrients. Instead, the liquid media acts as a bath for the cells, where they can freely absorb the nutrients they need and expel their waste back into the media. It may sound gross, cells sitting in their own dirt, but they are essentially doing that in our bodies as well. We just have a circulation system (our blood) that removes cell waste and filters it out, mostly using the liver and the kidneys. In fact, some of these waste products actually change the colour of the media which helps inform scientists when cells need to be fed again. Just like people, cells can be quite picky about their food, especially since they are surrounded by it, so different medias are available for different types of cells. For example, media to grow iPSC cells requires different types and concentrations of nutrients than media that grows blood cells. In fact, what nutrients a cell is surrounded by can influence how it behaves. For example, one of the common ways to determine if stem cells acquired from adult bone marrow still have stem cell-like properties, is to separately feed these cells with different types of media. For example, the bone marrow mesenchymal progenitor cells can become bone, cartilage, or fat, depending on the type of media used³. There is even specialized media used to can be used on iPSCs that cause cells to either grow into lung tissue or heart tissue. In both cases, even though the same initial cell is used, these different foods encourage stem cells to become a specific type of cell. Literally, cells are what they eat.

I: Bone marrow-derived mesenchymal stem cells (MSCs), II: Fat cells grown from MSCs, III: bone cells grown from MSCs, IV: Cartilage cells grown from MSCs

In order to feed cells, without getting them contaminated with things like bacteria or germs, sterile, isolated environments are required. The majority of cell culturing occurs in a culture hood. The culture hood acts like a workbench with controlled air flow to create a wind barrier in front of the opening where the scientist has to stick their hands through in order to do their experiments. This wall of air prevents particles and dust from entering the hood, as long as the scientist keeps their arms straight while they are working. As someone who has worked hours in such a position, it can be quite tough on the back, but great for posture! Before even working with the cells, the scientist has to make sure all their equipment and their workspace is sterile, which is most commonly achieved by spraying 70% ethanol on everything and everywhere, in addition to spraying it on the closed cell culture dishes themselves. Labs that do cell culture consume A LOT of alcohol (ethanol) 😉. Once the area is clean, scientists can bring their cell culture dishes into their workspace to perform a wide variety of experiments. For simple cell culturing to expand cell number, cell media is removed using a vacuum suction device that is often built into the hood. New media, warmed to 37˚C, is added manually using pipettes. Some cells float, such as blood (hematopoietic) cells, so they actually need to be spun really fast in a circle, using a centrifuge, to separate the cells from the media before removing the old media and adding new media. If the cells are running out of space in their dish, then they need to be “split”. Most cell types grow attached to the bottom of the culture dish, so it often requires the addition of an enzyme (such as trypsin) that will actively cleave proteins that allow the cells to stick ⁴. Scientists can see this happening when they look through a microscope. As cells detach from their culture dish, they will float around and look like little bubbles. It is at this point that they can be spun in a circle really fast in order to collect them into the bottom of a tube, and the redistributed into more culture dishes or collected for other experiments.

In actual practice, the lab coat would cover the arms so that there is no bare skin in the culture hood.

Cell culturing is a quintessential technique used in many basic medical science fields. Cells are the material that make up who we are and if they start misbehaving, this can lead to a myriad of diseases, including cancer. To understand how disease develops and how to treat them, it is good to start at the source, the cell. Cell culturing provides the material required to perform a wide variety of experiments without needing animal subjects. It is often involved in the first step of data collection to test a hypothesis and/or the feasibility of an experiment. Cell culture technqiues provide material to work with that allows scientists to perform a wide variety of experiments to better understand health and disease.

References

1. Al-Ani A, Toms D, Kondro D, Thundathil J, Yu Y, Ungrin M. Oxygenation in cell culture: critical parameters for reproducibility are routinely not reported . PLOS One 2018; 13(10):e0204269.
2. Price PJ. Best practices for media selection for mammalian cells . In Vitro Cell Dev Bio 2017; 53:673-681.
3. Ngo MA, Müller AL, Li Y, Neumann S, Tian G, Dixon IMC, Arora RC, Freed DH. Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts . Mol Cell Biochem 2014; 392:187-204.
4. Masters JR. Changing medium and passaging cells . Nature Protocols 2007;2:2276-2284.