Module 1: Cell Culture Basics

Photo by CDC on Unsplash

This is the first module in a four part series titled “Zero to One on the basic science behind cultivated meat”. You can click on these links to access: Course Overview, Module 2 (Cell Sources), Module 3 (Culture Medium), Module 4 (Process monitoring).

By the end of this module, you should understand:

  • Basic cell biology;
  • The difference between immortalized and primary cells;
  • The fundamental sterile cell culture techniques and equipment;
  • The basics of maintaining cell cultures: feeding, passaging, and cryopreservation.

All organisms can be classified as either eukaryotes or prokaryotes. This distinction is determined by the characteristics of the cells that make up these organisms. Watch this video on eukaryotic vs. prokaryotic cells (Youtube) to understand the differences.

Animals and plants are eukaryotes. The following article describes some of the eukaryotic organelles in more detail and has some excellent diagrams: Overview of eukaryotic cells (Lumen Learning).

  • Note: skip over the last section on comparing plant and animal cells.
An animal cell from https://courses.lumenlearning.com/boundless-biology/chapter/eukaryotic-cells/

This Overview of cells (Britannica.com) provides a more detailed description of fundamental cell biology concepts.

  • Due to the level of detail, it’s sufficient to just skim the content.
  • Don’t read the full article just yet — only read up to and including the section on the cell membrane, stopping at ‘internal membranes.’

Cell proliferation is a process that must occur in order to grow enough cells to make cultivated meat. Wikipedia has a good explanation of Cell proliferation (Wikipedia), which is the process of cells both growing and dividing.

To understand the cell cycle, watch this Cell cycle video (Youtube) which provides animations of how cells divide. The process of a cell copying itself and then dividing is called mitosis.

Study questions:

1.What are some of the key features that distinguish eukaryotic cells from prokaryotes?

2. How do nutrients (sugar, amino acids, electrolytes, and proteins) get inside the cell?

3. Which type of organelle is highly abundant in muscle cells, and why?

4. In the cell cycle, which phase do cells spend the majority of their time in?

Primary cells are extracted directly from tissues, such as the tissue biopsies that are used to start cell cultures in some cultivated meat companies. Read this page on Primary cell culture basics (Sigma-Aldrich/Merck) to understand some of the pros and cons of using primary cells, and how primary cells are different from cell lines.

Immortalized cells, also called cell lines, have been modified so that they can proliferate and be cultured indefinitely. Read this short piece on Immortalized cell lines (ScienceDirect) which provides a good overview of immortalized cells.

  • Only read the short article on ‘immortalized cell lines’ on the right side of the page — by Matt Carter and Jennifer Shieh 2015, 2nd edition.

For further reading, this article on Immortalized cell lines (Wikipedia) provides some additional detail and context on the topic.

Next, read this Immortalized cell culture guide (Creative Bioarray) to learn about the different ways that primary cells can be immortalized.

*Important note: Cell lines are typically immortalized cells, but this is not always the case. Some cell lines are derived from stem cells, which can be initially acquired as primary cells, and then propagated in culture for a long time (sometimes almost indefinitely, as is the case for embryonic stem cells, which are considered to be inherently immortal). For more information, read this article about Stem cell lines (Wikipedia). Cell sources are covered in more detail in Module 2.

Study questions:

5. What is cellular senescence?

6. What are some of the practical limitations of using primary cells?

7. For cultivated meat applications, would you choose to use primary cells or an immortalized cell line? Explain your reasoning.

8. What are some of the potential concerns that could arise from using immortalized cells to create food products?

Most cell types (both primary and cell lines) are adherent (adhesion-dependent), so they are typically cultured in 2D dishes or flasks, similar to the one pictured here:

The cells adhere to the bottom of the flask in a transparent monolayer that can only be seen using a microscope, and they are covered by enough nutrient-rich culture medium (the red liquid shown in the photo) to allow them to stay alive for a few days. The flasks are usually vented in order to allow oxygen into the flask, and the flasks are also left with plenty of air space inside them to allow enough oxygen to be transported to the cells. The flasks are usually made of plastic (polystyrene), which has been plasma-treated in order to permit cells to adhere to the surface. This type of treated surface is called ‘tissue culture plastic’ and is commonly abbreviated as TCP. You can read more about plasma treatment here (Harrick Plasma). The flasks are kept within an incubator designed to encourage cell growth, and removed from the incubator for regular monitoring and maintenance.

Read this brief Introduction to cell culture (Thermo Fisher).

  • Read the whole article, then watch the two videos at the bottom of the page:
  • Video 1: Introduction to Cell Culture
  • Video 2: Aseptic Techniques
  • Note: In the next section you will learn more information about cell confluence, subculturing, and cryopreservation.

This article lists out the essential Cell culture equipment (Thermo Fisher) that are necessary to have in a cell culture lab.

  • You can click on the links to learn more about the different pieces of equipment, but be sure to read over the page on Laminar flow hoods (Thermo Fisher). A class II laminar flow hood provides the best environment for working with cell cultures that do not pose a danger to the researcher.
  • Also be sure to read the page on Incubators.

Invitrogen has a helpful Cell culture basics handbook (Gibco/Invitrogen) that would be useful to save as a reference. For now, read this handbook through page 21, and just skim through the rest. We will go over cell culture maintenance in the next section.

Study questions:

9. What are the essential features of the artificial environment that are required in order to keep cell cultures alive?

10. Which chemical disinfectant is most frequently used during cell culture in order to clean your work area as well as your materials and equipment?

11. What types of biological contaminants might infect your cell cultures?

12. Is it recommended to use antibiotics in your cell cultures?

The easiest way to learn cell culture maintenance is from hands-on experience, however understanding the basic concepts and procedures beforehand will help you to adopt the proper techniques more quickly.

When maintaining cell cultures, the main way that you monitor your cells is by tracking their confluence. Watch this video: Cell culture to confluence (youtube).

Cell confluence (shown in the image below from Transfectopedia) is usually expressed as a percentage, which is typically determined by visually estimating how much of the cell culture dish is occupied by cells.

Cell Confluency images from https://www.mirusbio.com/transfectopedia/methods

As your cells are growing in their flasks, they must be checked regularly (ideally daily but at least every 2–3 days, depending on the cell type) to determine if they require maintenance. The most common forms of maintenance are feeding (changing the culture medium) and passaging, which must both be performed within a laminar flow hood. Passaging is also called subculturing or splitting the cells.

How do you know when to feed vs. when to passage?

  • Your cells should be fed if their confluency is still low (<80%) but they require a change of culture medium. You’ll know if the culture medium needs to be changed by the color, if your medium contains phenol red as a pH indicator. If phenol red is not used, you will instead follow a regular feeding schedule, usually every 2–3 days (depending on cell type and medium used).
  • Your cells should be passaged if their confluency is >80–90%. Passaging is performed in order to propagate your cells and to prevent crowding, which can cause your cells to change their behavior or die.

Feeding cells is simply changing the culture medium, but it still requires a strict sterile protocol that must be performed in the culture hood. Watch this Video on how to feed cells/change the media (Youtube).

  • Note: 70% ethanol is the standard recommended disinfectant, whereas in this video they use isopropyl alcohol.

Passaging cells is much more complicated because it involves detaching cells from the culture dish, centrifuging, counting, and transferring cells (at a lower cell density) to fresh culture dishes or flasks.

Read this web page on Passaging cells (Thermo Fisher), and this protocol on Subculturing adherent cells.

  • Also watch the video contained on the subculturing page (video: passaging cells).

Another useful Video on passaging primary cells (Youtube) shows this protocol again, but also has some helpful visuals that demonstrate what happens to the cells after the dissociation agent (trypsin) has been added.

Trypsin is the most commonly used dissociation reagent, and this page on cell dissociation with trypsin (Sigma-Aldrich) explains its usage. However, trypsin is derived from animal pancreas, therefore for fully animal-free systems, you’ll need to use a recombinant trypsin such as TrypLE.

An important part of passaging cells is counting cells before plating them into new flasks. If available, it is easiest to use automated systems such as the Countess (Thermo Fisher), but the cheapest option is to count them manually using a hemocytometer. Watch this video on cell counting to see how this is done.

Note about cell passaging: In typical biology labs, large numbers of cells are usually not required to perform experiments. Therefore when cells are split/passaged, only a few cells are transferred into new flasks, and the rest are discarded. For cultivated meat, all or nearly all of the cells are needed. Therefore during the process of cell proliferation, you will quickly end up with the situation shown in the photo below:

This image is from the New Harvest blog post on Mark Post’s cultured beef. This piece is worth a read to understand the challenge of scaling cell culture.

  • Note: these are 10-layer flasks, which permit the culture of 10 individual layers of cells within one (quite large) flask.

One strategy to avoid overloading your incubator (which can only accommodate so many flasks) is to regularly freeze down some of your cells, although this is best to do in early passages. Freezing your cells is called cryopreservation.

Cryopreservation should be performed fairly early on when propagating new primary cells, usually after just a few passages. The purpose is to store some of your cells so that you can then thaw and grow them at a later date. Read the following protocol on Cryopreservation of mammalian cells (Thermo Fisher) to gain an understanding of this process.Study questions:

13. What will happen to your cells if you let them reach 100% confluence?

14. How long should your cells be exposed to trypsin?

15. How can you determine if your cells are alive or dead?

16. How are frozen cell samples stored?

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