Tag: immortalised cell line

Textbook definition: The process by which cells are grown under controlled conditions. 

A cell culture is when cells that have been isolated from their original host are grown under artificial conditions. Cell culturing is one of the most important parts of bioprocessing, as without it there would be no bioprocessing!

We want to culture cells because cells are capable of performing complex reactions that might not be possible, or are economically unrealistic with chemical synthesis. Additionally, cells themselves can be commercially or medically desirable, such as stem cells. Here are some examples of what cell culturing can be used to make:

• Proteins (antibodies, hormones, gene therapy products)
• Vaccines
• Stem cells (for regeneration or replacement of organs or tissue)
• Biofuels
• Alcohol 
• Model systems for research

Model systems for research are cell cultures are used to test the effect of new drugs on living organisms. For example, testing the effectiveness of an antibiotic on a bacterial cell culture. Commonly used cell types for research model systems include e. coli cells, zebrafish cells, mice cells, pig cells and chimpanzee cells.

The advantages of using cell cultures are model systems for research are:

• Cells of interest can be observed without the interference of unwanted cells or tissue
• The effect on the cells can be monitored and observed in real-time because it is in vitro (outside the body) 
• Cheaper and faster than the majority of animal experiments
• Has far less ethical concerns than animal experiments
• In some cases can provide more representative data than using animal models
  

Types of cell cultures

Primary Cell culture

A primary cell culture is when the starting material has been extracted from tissue or organs of the host organism (i.e. via a biopsy). The culture has only been through a few population doublings and cannot be used indefinitely. Proteolytic enzymes are often used to digest the tissue into it’s single cell forms. 

Pros

• Representative of the original tissue in vivo (in the body) since it was recently extracted and hasn’t been mutated/ modified.

Cons

• Slow growth
• Short life-span – not many population doublings 
• Not well characterised – since the culture always comes from host tissue it is not well-understood, which makes it harder to grow efficiently

Secondary Culture

A secondary culture is formed after the primary cell culture undergoes its first subculture (the transfer of the cell culture to a new mediaL).

Established Cell line

If further subcultures are possible, an established or immortalised cell line is formed. As most cells stop dividing after a certain number of population doublings, to proliferate indefinitely one cell in the culture must undergo one of these changes:

• Random Mutation 
• Artificial modification (genetic engineering i.e extending the telomerase at the end of the cell’s DNA or insertion of a cancer causing gene, exposure to a cancer causing antigen i.e. radiation)

The oldest and most characterised human cell line is the HeLa Cell line, named after a woman called Henrietta Lacks. Henrietta died of cervical cancer in 1960, but before her death doctors extracted her cancer cells (without her knowledge). Cancer cells are able to undergo indefinite division and rapid proliferate due to a random mutation, and her cells formed the first immortalised human cell line.  This has helped achieve significant breakthroughs such as the creation of the polio vaccine. However, her family still has received none of the benefits and still cannot afford health insurance. Henrietta was a black woman, and for that reason doctors believed they did not need her consent or to give any recognition to her family, causing a lot of outrage and further implicates the need for ethics in the scientific field. The book, The Immortal Life of Henrietta Lacks, provides a great insight into the formation of bioethics and about the HeLa cell line, and I would recommend it if you have an interest in this field:

The Immortal Life of Henrietta Lacks by Rebecca Skloot. Click the picture to go to the amazon page!

Choice of cell:

The choice of what cell type to use depends on various factors:

• Source of organism: the ease of obtaining organism, the type of organism needed. For example, although embryonic stem cells are highly valued for their indefinite growth and regenerative capabilities, but as the organism is a human embryo it is a controversial source to use.
  
• Source of tissue: Ease of obtaining tissue, the type of cells needed – i.e. if testing effect of a medication on heart tissue you would not use neuronal cells.
  
• Primary culture or cell line: A cell line is usually preferred since they are more characterised, making it easier to grow them effectively and rapidly. However, for some cell types (i.e neuronal cells) only a primary culture is possible.

Some examples of common cell lines are E. coli, yeast, Chinese hamster ovary (CHO) cells

 Typical Growth Curve

We will go through growth curves in more detail in its own post, but I will outline what a growth curve should look like here. A growth curve represents the speed of proliferation of the cells, and tends to vary over the duration of a cell culture. There are 6 stages of cell growth:

1. Lag Phase: Cells have just been introduced to the media and are adapting to the new environment. Proliferation is close to 0.
2. Acceleration Phase: Cells begin to divide and growth rate starts to increase.
3. Growth Phase. The maximum growth rate possible is achieved. 
4. Deceleration Phase: Growth rate decreases over time due to a rate-limiting step (lack of nutrients, too little space)
5. Stationary Phase: Proliferation decreases to 0 due to rate limiting step. 
6. Death Phase: Cells begin to die due to rate limiting step. Growth rate turns negative. 

Graph depicting a typical growth curve.

The rate limiting step generally occurs when the nutrients, growth factors etc. in the media have been used up. To combat this subculturing is done. Subculturing is when the cell culture is moved to a fresh media, removing the rate limiting step and allowing the cells to re-enter the growth phase. This will be explained in more detail in the upstream processing section. 

Cell Quantification

Quantifying the cell culture is necessary to understand the growth of the cells and to ensure the cells are alive and proliferating. Cell culture quantification is usually done by cell counting. A viable cell count is more common as it tells us how many cells are alive, the proportion of alive to dead cells, etc. Cell counts are mainly automated these days, using a machine that stains the cells with a reagent such as tryphan blue, which only stains damaged cells, distinguishing the ratio of dead to alive cells.

This is just the basics of cell culture: we will go into cell culturing in more detail later on. If you have any questions or if I didn’t explain it thoroughly enough, please let me know down in the comments and I will give you more information and try to explain it with more detail. This is my first blog so I will appreciate any constructive criticism!