cell systems

Cell expression systems are the type of cell line used to express a protein or gene for commercial, research or medical use.

In bioprocessing, cells are used to create the desired product – proteins, antibodies, vaccines, etc. But there are so many different types of cell lines you can choose to use! Eukaryotic or prokaryotic, mammalian or fungi, human or animal.. theres a lot to choose from! Each cell line comes with its own advantages and disadvantages, and will be better for producing some products more than others.

In this post I will cover 3 main choices of cell line – prokaryotic (E. Coli), fungi (yeast) and mammalian, but in reality there are so many cell lines within those groups you can choose from! For example, Chinese Hamster Ovary, or CHO cell lines, are commonly used for producing monoclonal antibodies since they are mammalian cells, which are capable of complex protein folding and modification.

Prokaryotic: Escherichia Coli (E. Coli)

You’ve probably heard of E. Coli! E. Coli are a type of bacteria, and a very common cell expression system. This is because they are simple and well-characterised, since decades of research has been done using E. Coli as an expression system. Prokaryotic cell systems are simple in that they are unicellular, thus do not function as part of a larger organism. Prokaryotic cells also tend to proliferate at a rapid rate due to this (reproduction is carried out via mitosis). However, due to their simplicity the products they produce tend to also be very simple – protein folding and modification is very limited in these cells.

DNA integration and protein expression tends to be created by introducing plasmid vectors that carry the desired gene to the E. Coli cells. We will cover what plasmids are in more detail in a later post, but they are basically circular DNA that bacteria commonly use to transfer genes between one another. The plasmids can be engineering to carry the gene of interest (GOI) into the E. Coli cell, where it can then be acted upon by the cell’s genetic machinery to produce the desired protein.

E. Coli is used to produce a range of products, such as biofuels, vaccines and insulin! E. Coli can produce product titres (product concentrations) of up to 10 grams/ litre.

Pros:

Simple genetics (less DNA than other cell types)
Easy cultivation
Well-characterised: effective cultivation
Proven to be effective
Rapid growth and proliferation
Characterised media
Simple nutritional requirements

Cons:

No glycosylation of proteins
Inclusion bodies (condensed globs of protein)
Endotoxins
Not as similar to humans as other cell types
Limited protein modification
Fermentative metabolism (can proliferate without oxygen and produce toxic byproducts)

Eukaryotic: Fungi – Yeast

Yeast is another common cell expression system that you may have heard of. Yeast has been used to make many food products, including bread, wine and beer! As a eukaryotic cell, yeast is more similar to human cells than bacteria, and are capable of more protein modification than bacteria. However, yeast cells remain unicellular and can reproduce asexually, which makes it easier to grow them. As one of the first domesticated organisms in human history, yeast is very well-characterised, allowing for rapid and efficient growth.

Yeast cells are manipulated to produce more of the desired product via metabolic engineering. This is when pre-existing metabolic pathways in the cell are manipulated to create higher production of the desired product – for example a gene could be manipulated to produce more of the rate-limiting protein in a pathway that produces the desired product.

Yeast has been manipulated to produce many medicinal products such as insulin, antibody fragments and collagen. Product titres of yeast systems can reach 0.4 – 10 grams/litre.

Pros:

High cell density
Easy to grow
Characterised media
High product titre
Easy genetics
Product is secreted by cell
Simple nutritional requirements

Cons:

Yeast cells tend to over glycosylate the product
Possible toxicity
O2 mass transfer is hard with yeast cultures
Large amount of cell debris created
Complex downstream processing

Mammalian Cells

Mammalian cells are the most similar to human cells, and some cell lines are derived from human cells (look at HeLa cell lines in the Cell culture post)! Mammalian cells are eukaryotic cells, and are generally multicellular. Due to their similarity to human cells, products produced using mammalian cell systems tend to experience less rejection by the human body and are more effective. Complex mammalian proteins can also be made since mammalian cells are able to glycosylate and modify proteins the same way our cells do. However, due to their multicellular state reproduction (mitosis) can be limited – for example nerve cells tend to never undergo mitosis once matured. Also, mammalian cells have much slower growth.

Mammalian cell systems are manipulated into creating the desired protein via genetic engineering. This can cause either transient or stable expression. In transient expression, a vector (i.e plasmid) containing the gene of interest or mRNA of interest is introduced to the cell, where it is then acted upon by the cells genetic machinery to produce the product of interest. However since the gene is not incorporated into the host DNA it may degrade over a few generation doublings. In stable expression, the gene of interest is incorporated into the host cell’s DNA. This means the gene will be passed to future generations of the cell and will not degrade, so all cells produced from that cell will continue to express the desired product.

Mammalian cells are used to produce various products, such as monoclonal antibodies, growth hormones and interferon. Research is also being done to determine how to use mammalian cells to produce organ or tissue replacements. Product titres using mammalian cell systems are from 0.1 – 10 grams/ litre

Pros:

Product quality
Correct folding
Correct modifications
Potentially a simpler downstream process
Wider range of products possible

Cons:

Contamination risk
Slow growth
Not well characterised
Complex nutritional requirements
Long developmental time
Difficult scale-up
Safety concerns (use of viruses as vectors)
Potentially complex downstream process

How do you select what cell line to use?

When selecting a cell line, many factors come into play – price, developmental time, product quality, product yields, safety, purification, scaling of the process. This makes it difficult to select the perfect cell line to use for many products. However, ideally the cell line should meet these criteria:

Acceptable product quality and product quality attributes
Stable expression of the product throughout upstream processing
Desired yield of the product reached
Purification without loss of product quality

And there you have it! If you have any questions, leave a comment below and I will get back to you ASAP!