In vitro Reproducibility: Respect your cells!

In vitro Reproducibility: Respect your cells!

In recent years, there has been increased concern over the In Vitro reproducibility of observations in life science research, particularly in light of negative experiences with preclinical in vivo research. In vivo research is gradually being supplanted by cell-based systems, and the use of in vitro models is becoming more widespread. In the realm of in vitro biomedical research, strict criteria of In Vitro reproducibility and reliability must be created and maintained to avoid repeating past failures.

Keeping in mind the importance of reproducibility in cell culture, here is a list for you of the most vulnerable phases in every day in vitro cell culture routines that have the potential to affect cell quality and recommended procedures to reduce the likelihood of poor cell quality compromising reproducibility:

  • Selection of plasticware: Tissue culture polystyrene or polyethylene terephthalate (PET) are two materials used to make cell culture flasks and dishes. Each of these materials has unique surface qualities that influence how cells interface with the plastic surface. As a result of production variability, even similar shapes of cell culture dishes and plates made from the same type of plastic by various manufacturers can have variable surface qualities. Differentiation of cells can be significantly affected by the production and manufacturing of various types of plastics.
  • Cell Characterization: A significant portion of the research is done with a small number of tumor or immortalized cell lines. Sadly, the more unpretentious and widely distributed the cell line, the less emphasis is spent on its characterization. Long-term sub-culturing, for example, applies selective pressure on particular cells in the culture that develop faster.
  • Culture Media Selection: When a cell line is first introduced to a laboratory, the cell culture medium is often overlooked. It’s easy to ignore the fact that the electrolyte composition and carbohydrate content of such types of medium are often not suitable for physiological environments. An excessive amount of phosphate and/or a deficiency of calcium and magnesium are two common outcomes of using these media.
  • Cell Density and Change in Medium: Paracrine signaling and contact inhibition are more pronounced at higher cell densities. Cell metabolism is influenced by these events, which mediates cellular responses to pharmaceutical or toxicological compounds, and thus cell viability. It is clear that even minor differences in the total count of seeded cells, such as those caused by different counting methods, can result in huge differences in cell density after only a few days of expansion.
  • Mycoplasma Contamination: To avoid mycoplasma infections, it’s best to thaw and culture newly arrived cells in quarantine until the testing results arrive. It’s also crucial to avoid storing non-tested cells in liquid nitrogen tanks since direct physical contact between the cells and the nitrogen can happen. Furthermore, routine testing of applied cell lines should be performed multiple times each year to ensure early detection of contamination that may occur during normal activity.
  • Reporting: Insufficient, inadequate, and inaccurate reporting of methodology is a clear cause of poor reproducibility. Despite the fact that specific guidelines on GCCP and reporting requirements should be followed, a quick look at the materials and methods sections of submitted and published articles shows that there is still a long and tough path ahead.

The need for improved result reproducibility in the biomedical sciences has been acknowledged, and researchers are gradually internalizing it. Cutting-edge research is inextricably linked to a high proportion of false-positive findings. These false-positive results are a natural part of the research process and should not be confused with deliberate results manipulation.

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