Utilizing reduced serum media: an alternative approach to co-culture human dermal fibroblasts and epidermal keratinocytes

Atharva Bapat, Radhika and Snehal Kadam’s joint interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews.

Atharva Bapat is a 12th grade student at Suryadatta Public School, Pune. Atharva has a keen interest in Science and likes discovering new things by reading and experimenting. Atharva did a 3-week pre-college program on ‘Research Techniques in Biomedical Fields’ at Brown University, USA to learn first-hand about lab research techniques. He also represented India as part of his school team at the ASEP (Asian Student Exchange Program) conference in Kaohsiung, Taiwan and won the Platinum Award for best presentation. He has also participated in the COEP MUN (Model United Nations) as the delegate of Israel. Atharva is an achiever of the Maharashtra State Scholarship and few other prestigious competitive exams like Homi Bhabha Balvaidnyanik competition, Olympiads etc. A passionate football enthusiast, Atharva also recently started a podcast for football fans and loves to play football in his spare time. He has also captained his school and club football teams in the past. Being an avid traveller, Atharva likes exploring the world and has been to places like Iceland, Singapore, and Dubai.

Radhika is a senior research fellow at Institute of Bioinformatics and Biotechnology, University of Pune, and is developing human relevant, in-vitro platforms to study wound biofilm infections. She would like to study antibiotic resistance and find new ways to overcome it. She did her BSc in Biotechnology at Abasaheb Garware College, Pune and Masters in Biochemistry at University of Bremen, Germany. Her hobbies include sci-fi books and movies, and history trivia.

Snehal Kadam did her BS and MS degrees from IISER Pune, after which she worked as a research assistant with Dr. Karishma Kaushik for two years. Currently, she is a first year PhD student at the Hull York Medical School, United Kingdom. Fascinated by all things bacterial, she is particularly interested in understanding biofilms, their role in infections and host-pathogen interactions. She is also passionate about science communication, and runs the Talk To A Scientist program with her co-founder Dr. Karishma Kaushik. When not in the lab, she is probably out exploring the best food in town!

Here, authors talks about work titled “Reduced serum methods for contact-based coculture of human dermal fibroblasts and epidermal keratinocytes” published in Biotechniques.

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How would you explain your paper’s key results to the non-scientific community?

Human skin contains two major cell types, keratinocytes in the epidermis and fibroblasts in the dermis. Both cell types play important roles in wound infections and healing. It is challenging to grow these two cells together (also called co-culture) in the laboratory. Though both are part of the skin tissue, they have different serum requirements when cultured in the laboratory. Fibroblasts grow in the presence of 5-15% serum. Keratinocytes grow better under reduced serum or serum-free conditions. Moreover, both cell types require different growth factors. Recent work from our research group has found that reduced serum conditions can be used to successfully co-culture these two types of cells in the laboratory.

We have developed simple formulations which contain reduced concentrations of serum, to grow these two cell types together in the laboratory. The first formulation uses Minimum Essential Medium Eagle (MEM) along with Fibroblast Growth Supplement (FGS) and Keratinocyte Growth Supplement (KGS) (Figure 1A). The second formulation mixes equal amounts of a ready-to-use Fibroblast Growth Media (FGM) with ready-to-use Keratinocyte Growth Media (KGM) (Figure 1B). These supplements contain specific growth factors and nutrients needed for each cell type, and both the media formulations contain only 1-2% serum.

Figure 1

We observed that fibroblasts and keratinocytes grow together under both reduced serum media formulations. We showed that the two formulations can be used to grow the two cell types one over the other (layered approach) or together at the same time (co-seeding approach) (Figure 1C and D). In the layering method, keratinocytes are grown on top of the fibroblasts. This method mimics the layered structure of the skin. In the co-seeding method, both types of cells are added together at the same time. Co-seeding mimics the damaged wound, where the two cell types mix with each other. In these formulations, both cell types were able to attach, grow and proliferate under laboratory conditions. When we created a scratch in the cell grown together (to mimic a wound), the cells were also able to grow and migrate to close the wound (scratch) gap.

What are the possible consequences of these findings for your research area?

Many experiments in biology require cells isolated from human or animal tissues. In the laboratory, these living mammalian cells are kept in flasks containing nutrient-rich liquid medium and maintained at a constant temperature and carbon dioxide levels suitable for their proliferation. The animal-derived serum is widely used in cell culture media, in concentrations that range from 5-15% of the media composition. The most commonly used animal serum is often harvested from young cows and is known as Fetal Calf Serum (FCS) or Fetal Bovine Serum (FBS) (Figure 2).

Figure 2

Serum, which is separated from the blood itself, is composed of a range of growth factors, hormones, enzymes, minerals, and vitamins essential for the growth of most mammalian cells. When it is added to the medium, the cells in culture divide, grow, move, and display functions similar to that seen in the human body.

The use of serum has several ethical, technical and scientific concerns (Figure 3). 

Figure 3

Ethical concerns: The method of drawing blood from a foetal calf is harsh. It involves injuring the heart of an unborn foetal calf of a pregnant cow. Though special procedures are recommended to minimize pain to the foetus, these are often not strictly followed. Moreover, it is impossible to ensure that the foetus does not experience discomfort. One to three cow foetuses are required to obtain 1 liter of serum and approximately 2,000,000 bovine foetuses are injured each year to supply serum to research.

Scientific concerns: Various factors such as breeding conditions, food, and even animal-specific factors such as age and genetic profile, affects the composition of serum. In addition, treatments given to the cows such as antibiotics and hormones create fluctuations in different batches of serum. As a result, scientists are unsure of the exact composition of the serum. This can cause unexpected changes in the cells, lack of repeatability of experiments and misleading results. Animal sera may also contain microbes like mycoplasma and certain viruses, which are not only a major health risk for the researcher but may interfere with experiments.

Technical concerns: There is a high demand for good quality FBS in biological research. The quality control tests to detect toxins and microbes are rigorous and time-consuming. The imbalance between the production and supply of FBS has increased its cost, thereby increasing the cost of research that requires FBS.

The approach of reduced serum methods developed by our group enables the growth of two cell types with different serum requirements in the laboratory. It also reduces the ethical and scientific concerns with high concentrations of animal serum in cell culture. In doing so, this work leads to an important question, waiting to be explored – can this also be done for other cells?

The approach of reduced serum methods developed by our group enables the growth of two cell types with different serum requirements in the laboratory.

What was the exciting moment (eureka moment) during your research?

Snehal Kadam: The most eureka moment for me was when we were able to grow the two cell types (fibroblasts and keratinocytes) together in such low serum concentrations simultaneously. This had not been done previously in such a simple manner, and it was exciting to see that we were able to!

Radhika Dhekane: While working on this feature article, I realized the shocking extent of the animals used in research. However, it was good to learn that simple and promising solutions are available to reduce animal-derived products in the lab without compromising efficiency. 

Atharva Bapat: This was my first time working with a professional science team, and writing a science feature. I learnt a great deal about the topic as well as the writing process with my co-authors. Importantly, I learnt how to breakdown complex scientific concepts, to understand them and convey them to a broader audience.

What do you hope to do next?

In our group, we are working on in vitro, chronic wound infection platforms that recapitulate the wound microenvironment. Using these platforms, we are hoping to study the biology of chronic wound infection as well as perform high-throughput testing of antimicrobials. 

As we develop these platforms, we would like to explore ways to reduce the dependence on in vivo models by offering alternatives. We understand that it is difficult to replace available models completely. However, our approach is to build human-relevant platforms that bridge the gap between currently available in vitro and in vivo models. 

Where do you seek scientific inspiration?

Snehal Kadam: My inspiration comes from microbes! I find it fascinating that such tiny organisms can cause large-scale effects like infections in our body! I am also inspired by the amazing scientists who have mentored me through my journey in science.

Radhika Dhekane: The source of my scientific inspiration is the life happening at the microscopic level. More importantly, I am grateful for all the professors who have helped me to understand how exciting microbiology can be, not just from the textbooks, but also “live! from under-the-microscope!”.

Atharva Bapat: I get my scientific inspiration from nature and the things around me that I encounter daily. Each scientific article, textbook, or paper I read motivates me to learn even more about a particular subject. You can never have enough of science if you really enjoy it.

How do you intend to help Indian science improve?

In scientific research, several animals, including guinea pigs, zebrafish, frogs, mice, rats, hamsters, rabbits, dogs, and chimpanzees, are used as model systems. A model system is a simplified version of a biological organ or disease and gives scientists a system that is more accessible and amenable to experimentation.

It is increasingly being recognized that animal models do not perfectly mimic all aspects of human disease. For example, mice, commonly used to study skin wounds, poorly represent human wound healing. In mice, wounds close by contraction of muscle and tissue in their skin. On the other hand, human skin wounds heal by getting filled up with migrating cells from the edge of the wound, which progressively covers the wound area.

We are one of the very few labs in India working on inventing robust and human-relevant alternatives to animal models. By recognizing the scientific limitations and ethical concerns regarding the use of animals and animal products, we would like to bring the focus of Indian scientists to replacing and reducing such products in research.

Reference

Kadam S, Vandana M, Kaushik KS. Reduced serum methods for contact-based coculture of human dermal fibroblasts and epidermal keratinocytes. Biotechniques. 2020 Nov 1;69(5):347–55

Email: dhekane.radhika@gmail.com, snehalgkad@gmail.com, atharvabapat19@gmail.com

Lab: https://www.karishmakaushiklab.com/

Edited by: Sukanya Madhwal

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