CASE STUDIES

The Research Institute for Farm Animal Biology (FBN) in Dummerstorf, Germany is focused on exploring better livestock management within the context of sustainable farming. The Institute organizes its research into four main topics: individualising farm animal husbandry, farming animals in sustainable resource cycles, coping with critical life phases of farm animals, and promoting diversity in animal farming. Within this, a number of Working Groups explore specific themes. FBN’s Working Group Cell Biology of Muscle Growth is headed by Dr. Monika Röntgen. She outlines how new research tools, such as PHCbi’s LiCellMo, offer greater accuracy and timesaving capacity measuring important parameters in myogenesis (muscle development and growth).

Postnatal myogenesis depends on muscle stem cells including satellite cells (SC). The number and functional properties of these stem cells determine muscle formation, growth, regeneration potential, and adaptability. And, during critical periods of muscle development, their functionality can be changed permanently by a variety of factors.

“Our research aims to contribute to a better understanding of the processes of muscle development and plasticity towards improving muscle health, body composition, and meat quality in farm animals. We are also looking at alternative and environmentally friendly ways to produce animal protein as a component of global food security,” she explained. “Our main research topics are the molecular and metabolic control of early postnatal myogenic processes in farm animals, modulation of SC growth behaviour by dietary compounds, bioactive substances and components of the immune system, and generating protein for human nutrition from animal stem cells.”

Focused from the start

The work group is particularly interested in better understanding muscle development in the very early postnatal period. This is a critical period because two things happen. Firstly, the (adult) stem cell population develops. And secondly, the muscle tissue, in particular the myofibers, grow very rapidly.

“So far, not much research on the role of stem cells in early muscle development of farm animals has been carried out by others,” said Dr. Röntgen. “We focus on research in the pig because it is a very important farm animal, and is also a very interesting model for human medicine.”

“Specifically, we are trying to find out more about an important genetic disease that occurs in this early period: ‘splay leg syndrome’, which renders piglets unable to stand and, therefore, unable to drink. It can result in the early death of affected piglets,” she continued. “The pathogenesis of this disease is not really understood. It also occurs in many other species: humans, dogs and sheep. Increasing our understanding of the disease elucidates more about early and prenatal muscle development – both of which are disturbed in this disease.”

The Cell Biology of Muscle Growth Group is also very interested in finding out new ways of reducing the use of antibiotics through alternatives and concentrate on studying the role of probiotics in this.

Cultured meat

However, the Group’s biggest project at the moment is to develop a complete process to make cultured meat using pig stem cells. This project is funded by the German Federal Ministry of Education and Research and the FBN works together with three other partners: two research institutes and one company.

Scaling up

In Dr. Röntgen’s group, among others, M.Sc. Tessa Wolter works on this project, which is focused on finding solutions to produce high quality animal proteins in a cellbased way.

“In general, we are very interested in metabolic processes and the regulatory role of metabolic and mitochondrial signals in cell proliferation and differentiation, of which lactate plays an important role “, said Dr. Röntgen. “To produce muscle cells for cultured meat, we need to reach a higher efficiency of myogenesis. On way to do this is by changing the media composition in a way that can make the metabolism of the cells more effective.”

The LiCellMo can be used to analyse media samples taken from your bioreactor for glucose and lactate to control cell growth and metabolic activity.

“In our work, we have to perform a lot of tests to optimise our media with respect to glucose and other components,” she continued, “The LiCellMo helps us because we can see resulting positive or negative effects of such changes directly, in real-time. Normally, you would have to finish your experiments before you could start analysing the media samples. And this, of course, is a very time-consuming process.”

With the LiCellMo, the research team can directly see how the cells metabolic activity reflected by pH, glucose and lactate will change and can make fast decisions on the optimal time for media changes.

“Another point is that we have to scale up,” remarked Dr. Röntgen. “This is one of the biggest challenges because in standard cell culture we normally use dishes or flasks with small volumes of 12 to 20 ml. When you start working with a bioreactor, the volume is much higher and you need to find out first which cell density is optimal.

This sounds simple but this is the main point for all the other steps that will follow. In our case, we switched first from a 12 ml dish into a smaller bioreactor which has a whole volume of 200 millilitres, and started to work at a higher scale level by using a bioreactor which has a volume of 20 litres. Of course, you cannot quickly transfer the situation from the dish into this high volume. Thus, you have to control what happens over time. The LiCellMo can be used to analyse media samples taken from your bioreactor for glucose and lactate to control cell growth and metabolic activity.”

Practical benefits

Practical benefits “One advantage of the LiCellMo is its small size. It fits very well into our incubator and doesn’t occupy too much space. So, there is still enough space to do other experiments in parallel,” said Ms. Wolter. “The display showing the data on time was also very helpful.”

“The LiCellMo is simple to use, and most important, it is very, very sensitive. Therefore, it is very important to have media blanks for all tested conditions to differentiate between cell activities and handling effects, e.g., by opening the incubator door. “Another advantage of the LiCellMo is that you can do the experiments in a low volume format which help to save resources like primary cells or media.” she pointed out. “In contrast to the 96 well format, the 24 well plate format used with the LiCellMo is not too small. This is important because in many cell types the ability to proliferate and to differentiate differs depending upon the size of your dish. You have to find out the optimal format but 24 well is a nice format to start with and cells often grow better than in smaller formats.”

The LiCellMo is simple to use, and most important, it is very, very sensitive

Dr. Röntgen added that commercially available test kits have previously used for measuring lactate in particular, but the LiCellMo brings significant plus points.

“Lactate is very important in muscle, of course.” she said. “Typically, measuring lactate dehydrogenase activity in muscle tissue or cells is a standard method to get information on the metabolic situation. Another method is measuring the extracellular lactate concentration using test kits which in our hands were not sensitive enough specifically when working with cells. In addition, for both methods samples are analysed after finishing the experiment. Here, I must say, that the lactate analysis with the LiCellMo works much better because of its higher sensitivity.”

“It's also an advantage that you can carry out other experiments in parallel because the LiCellMo uses normal, commercially available cell culture plates,” Ms. Wolter said. “And, most important, you can use the cells for downstream investigations after monitoring them. For example, you can determine cell numbers, DNA and protein concentrations to characterise the cell growth or investigate the expression of genes to understand regulatory processes. It’s not an end point assay with the LiCellMo. If you are interested in other tests or treatments during the measurement, you can pause the LiCellMo at any moment and then continue measuring later on.”

Future potential

For Dr. Röntgen, direct measurements from larger bioreactors are likely to be key.

“It is already an advantage that we can analyse media from our bioreactors with equipment such as the LiCellMo,” she said. “We know that later on…really in the future…we will have much larger bioreactors, and then, it would be great to have a direct possibility to sample and control the media in a bioreactor over time. So that we can directly take a sample, analyse it, and get relevant information.”

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Find out more about the Research Institute for Farm Animal Biology (FBN)
https://www.fbn-dummerstorf.de/en/

Learn more about Dr. Monika Röntgen’s group:
https://www.fbn-dummerstorf.de/en/research/working-groups/cell-biology-of-muscle-growth/

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