Breaking the mould with jellyfish

Collagen is the most abundant protein in the human body and constitutes to over 25% of protein mass, it quite literally holds us all together.

It is a major component of the extracellular matrix, where its function is to provide tensile strength, regulate cell adhesion and migration, support tissue repair, and direct tissue development.

To date, at least 28 different collagens have been identified, these are classified according to the combination of three (out of a possible 46) polypeptide chains – called α chains ­­– that make up their structure. For example, collagen I is a heterotrimer comprised of two α1(I) chains and one α2(I) chain, while collagen II is a homotrimer made up of three α1(II) chains. The different collagen family members vary considerably in terms of tissue distribution, expression level, and the role they perform.

The role of collagen in cell culture
As the test subjects of research, cells have been the key ingredient for almost every medical advancement in the last century. 

Cell culture is a very important biological process that enables the testing and synthesis of all sorts of useful organic material. However, successful cell culture requires a highly specific environment that is precision-engineered to give the target cells the best possible chance of growing and developing. Collagen is often used as a culture media or substrate as it can create environmental conditions that are stable, nurturing, adaptable and beneficial to all sorts of cells.

Cells didn’t evolve to live in a lab, they prefer for example, the body’s extracellular matrix, the three-dimensional network of collagen, enzymes, and glycoproteins that give cells their unique structural and biochemical support.

Collagen provides a number of distinct advantages in cell culture over alternative substrate options such as laminin, synthetic peptides, plastic or sarcoma secretions:

• It has the ability to reproduce its inner environment so as to provide a structured medium for cells to interact with.
• It is biologically active and able to promote cell growth and adhesion. The collagen surface contains ligands that can bind target cells providing stability to samples and facilitating the delivery of nutrients.
• It is biodegradable, so host cells in the culture can replace collagen cells and build a bespoke extracellular matrix.
• It has a porous surface with gaps that are wide enough to enable other cells to migrate through it, yet small enough to facilitate their attachment to the collagen medium.
• Its unique structural properties make it suitable for 3D cell culture – which is increasingly important in the field.
• It doesn’t contain any chemicals or organic materials that are harmful to or cause allergic effects in humans, so analysts can handle it safely.

Collagen sources
The reliable and studied properties of these collagen types makes them invaluable to researchers, who require substrates to be a constant variable over multiple experiments. A simple change in pH or ionic concentrations between collagen batches can nullify an entire research project. Such batch-to batch inconsistency is often due to poor production and storage, but even when these are tightly regulated, the source of the collagen itself can create inconsistency. Complex organisms such as cows, pigs and rats can vary in age, sex, size, levels of fitness all of which can lead to batch-to-batch inconsistency in collagen products.

Here we take a look at the most popular collagen sources currently available to researchers;

Bovine
For decades, cows have been the prime industrial source of collagen. Plentiful, manageable and easy to breed, bovine collagen products have transformed reconstructive surgery and as a culturing bio-material, advanced global medical research. Collagen types I, II and IV can be sourced from the animal’s Achilles tendon, nasal cartilage and placental villi, respectively. However, bovine collagen’s successful status has not come without criticism. As a fellow mammal, cows can transfer multiple viruses and diseases to humans that non-mammalian sources cannot; outbreaks of transmissible spongiform encephalopathies (TSEs) and foot and mouth disease (FMD) continue to be reported in bovine collagen producing countries. Consequently, many researchers are looking for safer alternatives.

Aside from the disease risk, it has been estimated that approximately 3% of the population experience allergic reactions to bovine collagen. When the environmental sustainability of the meat industry is also taken into consideration, it is easy to understand why many lab professionals are seeking alternatives to bovine sources.

Porcine
Unlike bovine-collagen, porcine sourced proteins do not cause a significant allergic response in humans, perhaps owing to a closer homology to human collagen. Thus, the dermis and small intestinal mucosa of pigs have been widely used for biomaterial, tissue repair and cosmetic purposes. But, just like bovine collagen, porcine sources also come with a risk of disease and contamination. As a mammalian relative, the bacterial and viral pathogens of pigs are often transferable to humans, as most infamously demonstrated during the Swine Flu pandemic of 2009-10. As a pig product, porcine collagen is a prohibited substance for the nearly two billion people who follow certain religious orthodoxies. And, just like bovine products, pig derived collagen is sourced from a relatively complex organism that can have multiple variables and lead to batch-to-batch inconsistency and unpredictable collagen yield.

Rat tail
While bovine and porcine collagen serve the larger biomaterials industry, on the smaller, laboratory scale, rat tails are the preferred source for type I collagen. Due to its high accessibility and homogeneity, the animal’s protein is now the dominant source for laboratory slides, cover slips and gels. But there is a good reason that rat tail collagen is not as popular with the larger industry: its low yield and poor immunogenic profile. With its commercial collagen content limited to just its tail, the animal can hardly match the yields of its mammalian contemporaries. And when its structural fragility is considered, it is no wonder that industry projects tend to avoid rat tail collagen completely. The process of extracting rat tail collagen is sometimes done manually by labs and the quality control that is implemented can be questionable.

Recombinant collagen
As the dangers and limitations of animal-derived collagen become clearer, many alternative sources are rapidly gaining interest, with recombinant human collagen potentially providing a viable method of producing homogenous, batch to batch consistent collagen. However, collagen produced this way is the product of genetic engineering, and questions arise around both stability and achieving the correct morphology.

Recombinant collagen has been produced from both prokaryotic (E.Coli) and Eukaryotic (Yeasts and  Plant) based systems. These systems are scalable given the rapid growth of the organisms used and will ensure homogeneity across batches. However, prokaryotes and yeasts lack hydroxylases, which are required to form and fold the collagen into the required shape.

Plant  
Now used for the treatment of various chronic and infectious diseases, plant-derived collagen is quickly gaining a reputation for being economical, scalable, and safer than its bovine contemporaries. However, as an un-natural source, plant collagen has its limitations. Most plant-derived scaffolds form weaker and smaller fibre diameters than bovine materials and due to its farmed nature, production is at the mercy of environmental conditions such as droughts and blights. Thus, compared to animal-derived collagen, the market for plant derived collagen is expected to increase at the slowest rate until 2025, growing to just $46.5 million com-pared with animal market’s value of $480.5 million.

A collagen for now and the future
Major advances in disease therapies and regenerative medicine are on the horizon. But to reach them, researchers need a consistent, disease-free substrate that stimulates growth in all cell types. Considering the constraints of many collagens, to many this may seem unfeasible. But there is a solution.

Marine animals have been found to be the safest and one of the most applicable sources for obtaining collagen there is. Due to their safety and high natural collagen levels, animals such as fish, starfish, sponges and squid have been welcomed as exciting new collagen candidates. But one marine species has a collagen content potentially different to any other: Jellyfish. Thanks to collagen contents exceeding 40% in certain species, evolutionary ancient lineage, no risk of BSE/disease vector transfer and a compatibility with every common collagen type, jellyfish-derived collagen is set to reshape the biomaterials industry.

Cells in culture need supportive, nurturing substrates to promote growth and adhesion. This structure must also suit a cell’s unique morphology and requirements. Traditionally, these different needs were served by the common collagen types, I, II, III, & IV sourced from mammals. But thanks to its evolutionary conserved structure, jellyfish possess the ultimate precursor collagen sharing structural similarities to mammalian collagens – a highly flexible surface, able to act like all its mammalian collagen descendants rolled into one, without posing a disease risk.

Culturing cells is hard work. To replicate a cell’s unique environment requires a deep understanding of its needs and the right substrates to meet them. Can this collagen provide attachment? Can it allow for migration? Will it help the cells proliferate? These are just some of the many questions one must ask before choosing a biomaterial. And in recent times, researchers have had to ask themselves some more troubling questions, too. Will this collagen provoke an unwanted immune response? Will this bovine substrate transfer BSE? Are these materials safe for human use? When the tissue engineering field is poised to revolutionize regenerative medicine and stem cell therapies, these are not the questions the industry should be asking itself. Instead, the tissue engineering field must ask itself a bolder question: what is the future of collagen?

Many studies have shown that jellyfish collagen is able to replicate the cell adhesion, proliferation and migration that mammalian collagen is known for. But marine scaffolds do not just copy their mammalian counterparts. In many instances, they improve upon them. One study shows that Jellyfish collagen scaffolds provide an optimal in vitromicro-environment for ovarian cancer cells. The scaffold supports ovarian cancer cell proliferation, morphology and epithelial to mesenchymal transition markers.¹

And in another, investigating cortical neurons grown in co-culture with astrocytes showed that the mean correlation of the neuronal network synchronisation is higher on Jellagen compared to synchronisation on the current industry standard Matrigel², findings that could help advance the fields or drug discovery, tissue engineering and regenerative medicine.

While meeting and exceeding other collagens’ properties is important to researchers, no factor is more important than safety. When so much of the research that requires collagen is conducted on human tissue, with the aim of improving human health, there is no room for unsafe sources, such as those that harbour BSE or provoke an unwanted immune response in patients. Instead, the biomaterials industry must search for safe, disease-free sources that can give lab professionals the sanitary assurance they need, sources like jellyfish. As an ancient marine animal with low immunogenicity, jellyfish offer a safe, biocompatible collagen material, ideal for human implants thanks to a lower risk of immune reactions. Coupled with its safety, jellyfish collagen’s sustainability only furthers its value in the modern age of bio-materials. As the meat industry is considered one of the major contributors of CO₂ emissions, many researchers now long for a more responsible source. Again, jellyfish collagen can answer these calls for change.

Due to human marine activity, jellyfish blooms now blight coastal regions, causing dangerous problems for both marine life and human activity. But from its Cardiff base, Jellagen’s operations remove these growing pests and turn their abundant collagen into valuable, biocompatible cell culture products.

Authors:
Sophie Escott-Morgan is Senior Scientist Design & Development at Jellagen and Adam Watts is Digital Marketing Manager at Jellagen

www.jellagen.co.uk