Introduction
Stem cells are characterized by the unique abilities of self-renewal and differentiation, making them a powerful field of research for many applications (Hong et al., 2011). Fish stem cells are gaining popularity in the last few years thanks to the increasing interest in cellular aquaculture as an alternative and potentially more sustainable protein source to fishing. Currently, two of the main challenges, in developing cell-based seafood, are the lack of available fish cell lines and the poor knowledge about fetal bovine serum (FBS) substitutes for cell cultivation (Goswami et al., 2022). Here, we evaluate the effects of serum reduction on the proliferation of an Atlantic sturgeon cell line (AOXlar7y) derived from larval tissue (Grunow et al., 2011).
Materials and methods
Cell culture
For these experiments, Atlantic sturgeon stem cells from passages 43 to 47 and 78 to 83 were cultured in L-15 medium (Gibco) with 10% of fetal bovine serum (PAN Biotech) at a temperature of 25 and 28°C for proliferation analysis. All experiments were performed within six replicates using different passages.
Real-time cell electronic sensing
To evaluate the effect of lower serum concentration on cell growth, the impedance of the sturgeon stem cells was determined at 2%, 5%, and 10% serum concentration for 7 days. Cells were incubated at 25° C and 28°C in a 96-well plate and cell behaviour was measured via Cell impedance (CI) using the xCELLigence RTCA SP instrument (Roche Diagnostics GmbH). The CI is influenced by three cell parameters: cell number, cell adhesion, and cell size/morphology and can be seen as an indicator of proliferation.
Cell confluence
Cells were seeded in two 12-well plates (TPP) at a concentration of 70,000 cells per well and with three different serum concentrations (2%, 5%, 10%) and incubated for 2 days at 25 and 28°C. Cell confluence was evaluated using the Cell and Gene Therapy Catapult Cell Confluency Tool (https://ct.catapult.org.uk/resources/confluency-tool). Data were obtained from six microscope pictures per well which were taken on the third day of cultivation. Mean ± SD was analysed from each experimental trial.
Actin Filament staining
The morphology of AOXlar7y cells was evaluated by immunofluorescence labelling staining against β-actin. Cells were seeded on 3-chamber slides (Ibidi) at a concentration of 70,000 cells per chamber and cultured for 2 days at 25 and 28°C at 2%, 5% and 10% FBS concentration. For staining, fixed and permeabilised cells were stained with Phalloidin-iFluor 594 (Abcam) and DAPI (Carl Roth GmbH + Co. KG).
Results and Discussion
In this study, we evaluated the effect of serum reduction from 10% to 5% and 2% via real-time cell impedance measurement. The results show that a concentration of 5% FBS does not affect AOXlar7y cell proliferation. The curve from 5% is comparable to that of cells cultured with 10% of FBS, which is the standard amount of serum commonly added in most cell cultures. The proliferation curve of cells cultivated with 2% of FBS is instead markedly lower, confirming the direct correlation between serum quantity and cell proliferation capacity. The effect of the three FBS concentrations was further confirmed by comparing the cell confluence of each condition, which indicated an increase in proliferation for cells cultured at the higher temperature (28°C). The actin filament staining has been used to visualize the cytoskeleton to detect signs of stress or differentiation due to temperature increase and/or serum reduction. In particular, an abrupt serum starvation can be a stimulus for the stem cells differentiation (Messmer et al., 2022), making it more difficult to reduce serum during common stem cell maintenance. The results showed no evidence of differentiation, and cytoskeleton structures remained largely homogeneous between cells under different conditions. However, the cytoskeleton labelling occurred only after two days of cultivation, so further analysis is required after long-term culture.
Conclusions
The results show the possibility to lower the serum concentration for culturing the Atlantic sturgeon cell line AOXlar7y to 5%, without compromising their self-renewal capacity and without showing signs of stress induction or differentiation. By further investigating the properties of this cell line and the mechanisms by which it responds to certain culture conditions, it may allow us to determine the characteristics that make certain cells more proliferative even in the presence of less nutrition-rich, consequently cheaper, and more sustainable cultivation media. This principle is important not only for in vitro meat production, but also for general laboratory research, as it would make these powerful in vitro models more accessible worldwide.
References
Goswami M., Shambhugowda Y.B., Sathiyanarayanan A., Pinto N., Duscher A., Ovissipour R., Lakra W.S. and Nagarajarao R.C. (2022) Cellular Aquaculture: Prospects and Challenges. Micromachines, 13, 828
Grunow B., Noglick S., C. Kruse, M. Gebert (2011) Isolation of cells from Atlantic sturgeon Acipenser oxyrinchus oxyrinchus and optimization of culture conditions. Aquat Biol, 14: 67–75
Hong N., Li Z., Hong Y. (2011) Fish Stem Cell Cultures. Int. J. Biol. Sci., 7(4):392-402
Flack J.E. (2022) A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation. Nature Food volume, 3, 74–85