In the last six months the major experimental output has been centered around the testing of several recombinant growth factors for their use in a serum-free cell culture media. More than 30 different growth factors across six growth factor family types and based on protein sequences from 20 very different and exotic species of animals was screened in seven different concentrations. These recombinant growth factors were produced inexpensively in e. coli by Canadian collaborators. After initial screening of optimal concentration ranges the challenging experimental setup of combining many different components have begun. By the use of design of experiments (DOE) and response surface methodology (RSM) approaches this complicated multi-factorial serum-free media optimization has been made more efficient and experimentally feasible to perform. Below is a result from a RSM experiment showing the optimal concentrations of two factors (FGF-2 growth factor and fetuin, a carrier protein).
Currently, a 3-month research stay in Norway at the food research company Nofima is being undertaken. Here the focus is on how recombinant FGF-2 growth factors activate cell proliferation by analyzing signaling pathways and downstream gene expression.
Other than the work on growth factors the last six months, the isolation of bovine satellite cells from three different young animals (~2-month-old) has been performed. This will enable an in-depth comparison of old versus young bovine satellite cells. Excitingly, a new piece of equipment has also been introduced into the cell lab; the multi-modal cell imager Cytation 5. The process of setting up and optimizing protocols have been started and will eventually streamline many important cell characterization procedures such as proliferation assays and myotube quantification. This machinery will enable several high-throughput and automatic image-based techniques which will support the cell-based research significantly. Below is a graphical abstract showing how the Cytation 5 will be a central part on the ongoing research.
The main focus of 2021 will be the optimization of a serum-free growth media, mainly by testing different recombinant growth factors. The graph below demonstrates the great effect of animal serum in the cell media, which will be difficult to entirely replicate. Another topic that will be investigated is how the age of the donor cow affects the performance of the satellite cells isolated.
Different myo-satellite cell isolation strategies have been performed with success. The above picture is an example of the tissue explant method where cells are expanding from a piece of muscle tissue. Isolation of viable cells after several days of tissue storage has been demonstrated as well.
A protocol for the visualization of satellite cell fusion and myo-fiber formation has been developed. Also a software-based quantitative analysis of these fusion images has been implemented. The expression of certain myogenic markers was investigated using qPCR. The myotubes showed here are from satellite cells isolated after 5 days of muscle tissue storage.
