Department of Basic Medical Sciences, Wroclaw Medical University, 50-556 Wroclaw, Poland.
Department of Toxicology, Wroclaw Medical University, 50-556 Wroclaw, Poland.
Cells. 2020 Apr 14;9(4):958. doi: 10.3390/cells9040958.
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.
PC12 细胞系是神经科学研究中最常用的细胞系之一，包括神经毒性、神经保护、神经分泌、神经炎症和突触发生研究。ATCC 收藏中有两种类型的 PC12 细胞系：悬浮培养的传统 PC12 细胞和贴壁良好的贴壁表型。悬浮培养的 PC12 细胞往往会聚集，并且不易附着在非涂层表面上。因此，有必要修饰培养容器的表面。本文旨在描述两种不同变体的 PC12 细胞的使用情况，并描述它们在不同 NGF 浓度（大鼠或人源）下在各种表面上的分化和神经元生长。在我们的研究中，我们评估了细胞形态、神经突长度、密度和生长（通过分光荧光法测量）以及神经元生物标志物（双皮质蛋白和 NeuN）的表达。我们发现胶原蛋白涂层是两种细胞系最通用的表面修饰方法。对于贴壁细胞，涂层的重要性肯定较低，而多聚-d-赖氨酸表面与胶原蛋白一样好。我们还证明了 NGF 的浓度对细胞分化程度非常重要。对于悬浮细胞，我们在 100 ng/mL NGF（每 48 小时更换一次）孵育 14 天后获得了最佳的神经元特征（神经突的长度和密度），而对于贴壁细胞，在 3-5 天后开始增殖。在 PC12 细胞系中，在细胞质中发现双皮质蛋白（DCX）表达，在细胞核中发现 NeuN 表达。相反，在 PC12 Adh 系中，未检测到 DCX 表达，而 NeuN 表达位于整个细胞（核和细胞质中）。只有用 NGF 分化后的悬浮培养的传统 PC12 系才应用于神经生物学研究，特别是在 NeuN 蛋白的作用得到充分理解之前，该蛋白的表达也在贴壁细胞的细胞质中被注意到。
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