What you need to know about cells – Bioprocessing Explained

Cell expression 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.

Cell Systems

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!

Cell culture

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:

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

typical growth curve of a cell culture, with stages lag, acceleration, growth, deceleration, stationary and death

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!

Cells: A quick recap

I’m sure most of you will know this from high school, but I thought I’d go over it anyways as you’ll probably get tested on this. I’ll make a section on the molecular biology aspects of bioprocessing if this blog gets popular enough for people to request it, though as molecular biology is much more popular of a subject you can definitely find other bloggers who have much more knowledge about it than I do.

Cell: Textbook definition: The smallest structural and functional unit of any living organism, capable of growth, reproduction, functional activity and changes before death.

There are 2 main types of cells: Prokaryotic cells and Eukaryotic cells. Below is a list of differences between them.

Prokaryotic Cells	Eukaryotic cells
Circular DNA	Linear DNA
No organelles, just sub-compartments	Membrane enclosed organelles
70S ribosomes	80S ribosomes
Nucleus absent – naked DNA	Nucleus present
Cell division via fission or budding	Cell division via mitosis
1 chromosome	More than 1 chromosome
Cell wall, made of peptidoglycans	If there is a cell wall, made of chitin or cellulose
Generally smaller	Generally bigger

Those are probably the most important differences between them. Bacteria are prokaryotes, mammals, plants and fungi are eukaryotes.

Now for organelles: Organelles are basically the organs of the cell. These are only present in eukaryotic cells, except for ribosomes. Prokaryotic cells perform reactions in the cytoplasm and with the external environment. Below is a list of the organelles and functions:

Nucleus	Contains the DNA
Nucleolus	Within the nucleus, where ribosomes are synthesized
Ribosomes	Synthesize proteins
Mitochondria	Generate ATP i.e energy
Rough Endoplasmic reticulum (ER)	Has many associated ribosomes, site of membrane and secretory protein synthesis
Smooth ER	Makes lipids
Golgi Body	Modifies, packages and sorts proteins
Cytosol	Internal fluid of cell – area of cell signalling, protein synthesis, intermediary reactions
Cell membrane	Regulates transport of molecules in and out of the cell
Lysozymes	Contain digestive proteins

prokaryotic cell diagram — A prokaryotic cell – again, excuse the poorly made sketch, don’t wanna get sued

mammalian cell diagram — A mammalian cell – plant and fungi cells have cell walls.