Abstract
The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3–5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.
Highlights
Recent neurotoxicity tests on nervous system diseases rely mostly on animal models
In the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell
The traditional PC12 line grown in suspension after differentiation with nerve growth factor (NGF) should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has been noted in the cytoplasm of adherent cells, is well understood
Summary
Recent neurotoxicity tests on nervous system diseases rely mostly on animal models. These experiments are associated with ethical questions as well as are expensive, laborious, and often produce vague results [1]. On the other hand, are relatively fast and less demanding ways to test chemicals for their neurotoxic properties [1]. In vitro techniques to study neurodegeneration are based principally on chemical-induced effects in cell lines derived from rodent or human, and allow one to uncover basic, specific impacts on individual cells or relevant molecular pathways [2]. Proper design of the experiment, meaning the suitability of a cell model that will be optimal for responding to a particular hypothesis, relies principally on the right choice of the cell line. The use of an inappropriate model may lead to an incorrect assessment of neurotoxic
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